Novel anti-arrythmic and heart failure drugs that target the leak in the ryanodine receptor (RYR2)

ABSTRACT

The present invention provides novel 1,4-benzothiazepine intermediates and derivatives, methods for synthesizing same, and methods for assaying same. The present invention also provides methods for using these novel compounds to limit or prevent a decrease in the level of RyR2-bound FKBPl2.6 in a subject; to prevent exercise-induced sudden cardiac death in a subject; and to treat or prevent heart failure, atrial fibrillation, or exercise-induced cardiac arrhythmia in a subject. The present invention further provides methods for identifying an agent that enhances binding of RyR2 and FKBP12.6, and agents identified by these methods. Additionally, the present invention provides methods for identifying agents for use in treating or preventing heart failure, atrial fibrillation, or exercise-induced cardiac arrhythmia, and in preventing exercise-induced sudden cardiac death. Also provided are agents identified by such methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This present Divisional Application claims the benefit of U.S.Continuation-in-Part patent application Ser. No. 10/763,498, filed onJan. 22, 2004; which claims the benefit of U.S. Continuation-in-Partpatent application Ser. No. 10/680,988, filed on Oct. 7, 2003; whichclaims the benefit of U.S. Continuation-in-Part patent application Ser.No. 10/608,723, filed on Jun. 26, 2003; which claims the benefit of U.S.Continuation patent application Ser. No. 10/288,606, filed on Nov. 5,2002; which claims the benefit of U.S. patent application Ser. No.09/568,474, filed on May 10, 2000, now U.S. Pat. No. 6,489,125 B1,issued on Dec. 3, 2002; the contents of which are hereby incorporated byreference thereto.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under NIH Grant No. PO1HL 67849-01. As such, the United States government has certain rights inthis invention.

BACKGROUND OF THE INVENTION

Despite advances in treatment, congestive heart failure remains animportant cause of mortality in Western countries. Heart failure affects5 million individuals in the United States alone, and is characterizedby a 5-year mortality rate of ˜50% (Levy et al., Long-term trends in theincidence of and survival with heart failure. N. Engl. J. Med.,347:1397-402, 2002). An important hallmark of heart failure is reducedmyocardial contractility (Gwathmey et al., Abnormal intracellularcalcium handling in myocardium from patients with end-stage heartfailure. Circ. Res., 61:70-76, 1987).

In healthy heart muscle, and other striated muscle, calcium-releasechannels on the sarcoplasmic reticulum (SR), including ryanodinereceptors (RyRs), facilitate coupling of the action potential to amuscle cell's contraction (i.e., excitation-contraction (EC) coupling).Contraction is initiated when calcium (Ca²⁺) is released from the SRinto the surrounding cytoplasm. In heart failure, contractileabnormalities result, in part, from alterations in the signaling cascadethat allows the cardiac action potential to trigger contraction. Inparticular, in failing hearts, the amplitude of the whole-cell Ca²⁺transient is decreased (Beuckelmann et al., Intracellular calciumhandling in isolated ventricular myocytes from patients with terminalheart failure. Circ., 85:1046-55, 1992; Gomez et al., Defectiveexcitation-contraction coupling in experimental cardiac hypertrophy andheart failure. Science, 276:800-06, 1997), and the duration prolonged(Beuckelmann et al., Intracellular calcium handling in isolatedventricular myocytes from patients with terminal heart failure. Circ.,85:1046-55, 1992).

Cardiac arrhythmia, a common feature of heart failure, results in manyof the deaths associated with the disease. Atrial fibrillation (AF) isthe most common cardiac arrhythmia in humans, and represents a majorcause of morbidity and mortality (Chugh et al., Epidemiology and naturalhistory of atrial fibrillation: clinical implications. J. Am. Coll.Cardiol., 37:371-78, 2001; Falk, R. H., Atrial fibrillation. N. Engl. J.Med, 344:1067-78, 2001). Despite AF's clinical importance, the molecularmechanisms underlying this arrhythmia are poorly understood, andtreatment options are limited.

It is well established that structural and electricalremodeling—including shortening of atrial refractoriness, loss ofrate-related adaptation of refractoriness (Wijffels et al., Atrialfibrillation begets atrial fibrillation: a study in awake chronicallyinstrumented goats. Circulation, 92:1954-68, 1995; Morillo et al.,Chronic rapid atrial pacing: structural, functional, andelectrophysiological characteristics of a new model of sustained atrialfibrillation. Circulation, 91:1588-95, 1995; Elvan et al.,Pacing-induced chronic atrial fibrillation impairs sinus node functionin dogs: electrophysiological remodeling. Circulation, 94:2953-60, 1996;Gaspo et al., Functional mechanisms underlying tachycardia-inducedsustained atrial fibrillation in a chronic dog model. Circulation,96:4027-35, 1997), and shortening of the wavelength of re-entrantwavelets—accompany sustained tachycardia (Rensma et al., Length ofexcitation wave and susceptibility to reentrant atrial arrhythmias innormal conscious dogs. Circ. Res., 62:395-410, 1988). This remodeling islikely important in the development, maintenance and progression ofatrial fibrillation.

Previous studies suggest that calcium handling may play a role inelectrical remodeling in atrial fibrillation (Sun et al., Cellularmechanisms of atrial contractile dysfunction caused by sustained atrialtachycardia. Circulation, 98:719-27, 1998; Goette et al., Electricalremodeling in atrial fibrillation: time course and mechanisms.Circulation, 94:2968-74, 1996; Daoud et al., Effect of verapamil andprocainamide on atrial fibrillation-induced electrical remodeling inhumans. Circulation, 96:1542-50, 1997; Yu et al., Tachycardia-inducedchange of atrial refractory period in humans: rate dependency andeffects of antiarrhythmic drugs. Circulation, 97:2331-37, 1998; Leistadet al., Atrial contractile dysfunction after short-term atrialfibrillation is reduced by verapamil but increased by BAY K8644.Circulation, 93:1747-54, 1996; Tieleman et al., Verapamil reducestachycardia-induced electrical remodeling of the atria. Circulation,95:1945-53, 1997). However, regulation of RyR2 during atrialfibrillations has not previously been reported.

Approximately 50% of all patients with heart disease die from fatalcardiac arrhythmias. In some cases, a ventricular arrhythmia in theheart may be rapidly fatal—a phenomenon referred to as “sudden cardiacdeath” (SCD). Fatal ventricular arrhythmias (and SCD) may also occur inyoung, otherwise-healthy individuals who are not known to havestructural heart disease. In fact, ventricular arrhythmia is the mostcommon cause of sudden death in otherwise-healthy individuals.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is aninherited disorder in individuals with structurally-normal hearts. It ischaracterized by stress-induced ventricular tachycardia—a lethalarrhythmia that may cause SCD. In subjects with CPVT, physical exertionand/or stress induce bidirectional and/or polymorphic ventriculartachycardias that lead to SCD in the absence of detectable structuralheart disease (Laitinen et al., Mutations of the cardiac ryanodinereceptor (RyR2) gene in familial polymorphic ventricular tachycardia.Circulation, 103:485-90, 2001; Leenhardt et al., Catecholaminergicpolymorphic ventricular tachycardia in children: a 7-year follow-up of21 patients. Circulation, 91:1512-19, 1995; Priori et al., Clinical andmolecular characterization of patients with catecholaminergicpolymorphic ventricular tachycardia. Circulation, 106:69-74, 2002;Priori et al., Mutations in the cardiac ryanodine receptor gene (hRyR2)underlie catecholaminergic polymorphic ventricular tachycardia.Circulation, 103:196-200, 2001; Swan et al., Arrhythmic disorder mappedto chromosome 1q42-q43 causes malignant polymorphic ventriculartachycardia in structurally normal hearts. J. Am. Coll. Cardiol.,34:2035-42, 1999).

CPVT is predominantly inherited in an autosomal-dominant fashion.

Individuals with CPVT have ventricular arrhythmias when subjected toexercise, but do not develop arrhythmias at rest. Linkage studies anddirect sequencing have identified mutations in the human RyR2 gene, onchromosome 1q42-q43, in individuals with CPVT (Laitinen et al.,Mutations of the cardiac ryanodine receptor (RyR2) gene in familialpolymorphic ventricular tachycardia. Circulation, 103:485-90, 2001;Priori et al., Mutations in the cardiac ryanodine receptor gene (hRyR2)underlie catecholaminergic polymorphic ventricular tachycardia.Circulation, 103:196-200, 2001; Swan et al., Arrhythmic disorder mappedto chromosome 1q42-q43 causes malignant polymorphic ventriculartachycardia in structurally normal hearts. J. Am. Coll. Cardiol.,34:2035-42, 1999).

There are three types of ryanodine receptors, all of which arehighly-related Ca²⁺ channels: RyR1, RyR2, and RyR3. RyR1 is found inskeletal muscle, RyR2 is found in the heart, and RyR3 is located in thebrain. The type 2 ryanodine receptor (RyR2) is the major Ca²⁺-releasechannel required for EC coupling and muscle contraction in cardiacstriated muscle.

RyR2 channels are packed into dense arrays in specialized regions of theSR that release intracellular stores of Ca²⁺, and thereby trigger musclecontraction (Marx et al., Coupled gating between individual skeletalmuscle Ca²⁺ release channels (ryanodine receptors). Science, 281:818-21,1998). During EC coupling, depolarization of the cardiac-muscle cellmembrane, in phase zero of the action potential, activates voltage-gatedCa²⁺ channels. In turn, Ca²⁺ influx through these channels initiatesCa²⁺ release from the SR via RyR2, in a process known as Ca²⁺-inducedCa²⁺ release (Fabiato, A., Calcium-induced release of calcium from thecardiac sarcoplasmic reticulum. Am. J. Physiol., 245:C1-C14, 1983;Nabauer et al., Regulation of calcium release is gated by calciumcurrent, not gating charge, in cardiac myocytes. Science, 244:800-03,1989). The RyR2-mediated, Ca²⁺-induced Ca²⁺ release then activates thecontractile proteins which are responsible for cardiac musclecontraction.

RyR2 is a protein complex comprising four 565,000-dalton RyR2polypeptides in association with four 12,000-dalton FK506 bindingproteins (FKBPs), specifically FKBP12.6 (calstabin). FKBPs are cis-transpeptidyl-prolyl isomerases that are widely expressed and serve a varietyof cellular functions (Marks, A. R., Cellular functions ofimmunophilins. Physiol. Rev., 76:631-49, 1996). FKBP12 proteins aretightly bound to, and regulate the function of, the skeletal ryanodinereceptor, RyR1 (Brillantes et al., Stabilization of calcium releasechannel (ryanodine receptor) function by FK506-binding protein. Cell,77:513-23, 1994; Jayaraman et al., FK506 binding protein associated withthe calcium release channel (ryanodine receptor). J. Biol. Chem.,267:9474-77, 1992); the cardiac ryanodine receptor, RyR2 (Kaftan et al.,Effects of rapamycin on ryanodine receptor/Ca(2+)-release channels fromcardiac muscle. Circ. Res., 78:990-97, 1996); a related intracellularCa²⁺-release channel, known as the type 1 inositol 1,4,5-triphosphatereceptor (IP3R1) (Cameron et al., FKBP12 binds the inositol1,4,5-trisphosphate receptor at leucine-proline (1400-1401) and anchorscalcineurin to this FK506-like domain. J. Biol. Chem., 272:27582-88,1997); and the type 1 transforming growth factor β (TGFβ) receptor(TβRI) (Chen et al., Mechanism of TGFbeta receptor inhibition by FKBP12.EMBO J., 16:3866-76, 1997). FKBP12.6 binds to the RyR2 channel (onemolecule per RyR2 subunit), stabilizes RyR2-channel function (Brillanteset al., Stabilization of calcium release channel (ryanodine receptor)function by FK506-binding protein. Cell, 77:513-23, 1994), andfacilitates coupled gating between neighboring RyR2 channels (Marx etal., Coupled gating between individual skeletal muscle Ca²⁺ releasechannels (ryanodine receptors). Science, 281:818-21, 1998), therebypreventing aberrant activation of the channel during the resting phaseof the cardiac cycle.

Phosphorylation of cardiac RyR2 by protein kinase (PKA) is an importantpart of the “fight or flight” response that increases cardiac ECcoupling gain by augmenting the amount of Ca²⁺ released for a giventrigger (Marks, A. R., Cardiac intracellular calcium release channels:role in heart failure. Circ. Res., 87:8-11, 2000). This signalingpathway provides a mechanism by which activation of the sympatheticnervous system, in response to stress, results in increased cardiacoutput required to meet the metabolic demands of the stress responses.Upon binding of catecholamines, β1- and β2-adrenergic receptors activateadenylyl cyclase via a stimulatory G-protein, Gα_(s). Adenylyl cyclaseincreases intracellular cAMP levels, which activate the cAMP-dependentPKA. PKA phosphorylation of RyR2 increases the open probability of thechannel by dissociating calstabin2 (FKBP12.6) from the channel complex.This, in turn, increases the sensitivity of RyR2 to Ca²⁺-dependentactivation (Hain et al., Phosphorylation modulates the function of thecalcium release channel of sarcoplasmic reticulum from cardiac muscle.J. Biol. Chem., 270:2074-81, 1995; Valdivia et al., Rapid adaptation ofcardiac ryanodine receptors: modulation by Mg²⁺ and phosphorylation.Science, 267:1997-2000, 1995; Marx et al., PKA phosphorylationdissociates FKBP12.6 from the calcium release channel (ryanodinereceptor): defective regulation in failing hearts. Cell, 101:365-76,2000).

Failing hearts (e.g., in patients with heart failure and in animalmodels of heart failure) are characterized by a maladaptive responsethat includes chronic hyperadrenergic stimulation (Bristow et al.,Decreased catecholamine sensitivity and beta-adrenergic-receptor densityin failing human hearts. N. Engl. J. Med., 307:205-11, 1982). Thepathogenic significance of this stimulation in heart failure issupported by therapeutic strategies that decrease beta-adrenergicstimulation and left ventricular myocardial wall stress, and potentlyreverse ventricular remodeling (Barbone et al., Comparison of right andleft ventricular responses to left ventricular assist device support inpatients with severe heart failure: a primary role of mechanicalunloading underlying reverse remodeling. Circulation, 104:670-75, 2001;Eichhorn and Bristow, Medical therapy can improve the biologicalproperties of the chronically failing heart. A new era in the treatmentof heart failure. Circulation, 94:2285-96, 1996). In heart failure,chronic beta-adrenergic stimulation is associated with the activation ofbeta-adrenergic receptors in the heart, which, through coupling withG-proteins, activate adenylyl cyclase and thereby increase intracellularcAMP concentration. cAMP activates cAMP-dependent PKA, which has beenshown to induce hyperphosphorylation of RyR2.

Thus, chronic heart failure is a chronic hyperadrenergic state (Chidseyet al., Augmentation of plasma norepinephrine response to exercise inpatients with congestive heart failure. N. Engl. J. Med., 267:650, 1962)which results in several pathologic consequences, including PKAhyperphosphorylation of RyR2 (Marx et al., PKA phosphorylationdissociates FKBP12.6 from the calcium release channel (ryanodinereceptor): defective regulation in failing hearts. Cell, 101:365-76,2000).

The PKA hyperphosphorylation of RyR2 has been proposed as a factorcontributing to depressed contractile function and arrhythmogenesis inheart failure (Marks et al., Progression of heart failure: is proteinkinase a hyperphosphorylation of the ryanodine receptor a contributingfactor? Circulation, 105:272-75, 2002; Marx et al., PKA phosphorylationdissociates FKBP12.6 from the calcium release channel (ryanodinereceptor): defective regulation in failing hearts. Cell, 101:365-76,2000). Consistent with this hypothesis, PKA hyperphosphorylation of RyR2in failing hearts has been demonstrated in vivo, both in animal modelsand in patients with heart failure undergoing cardiac transplantation(Antos et al., Dilated cardiomyopathy and sudden death resulting fromconstitutive activation of protein kinase A. Circ. Res., 89:997-1004,2001; Marx et al., PKA phosphorylation dissociates FKBP12.6 from thecalcium release channel (ryanodine receptor): defective regulation infailing hearts. Cell, 101:365-76, 2000; Ono et al., Altered interactionof FKBP12.6 with ryanodine receptor as a cause of abnormal Ca²⁺ releasein heart failure. Cardiovasc. Res., 48:323-31, 2000; Reiken et al.,Beta-adrenergic receptor blockers restore cardiac calcium releasechannel (ryanodine receptor) structure and function in heart failure.Circulation, 104:2843-48, 2001; Semsarian et al., The L-type calciumchannel inhibitor diltiazem prevents cardiomyopathy in a mouse model. J.Clin. Invest., 109:1013-20, 2002; Yano et al., Altered stoichiometry ofFKBP12.6 versus ryanodine receptor as a cause of abnormal Ca²⁺ leakthrough ryanodine receptor in heart failure. Circulation, 102:2131-36,2000).

In failing hearts, the hyperphosphorylation of RyR2 by PKA induces thedissociation of the regulatory FKBP12.6 subunit from the RyR2 channel(Marx et al., PKA phosphorylation dissociates FKBP12.6 from the calciumrelease channel (ryanodine receptor): defective regulation in failinghearts. Cell, 101:365-76, 2000). This causes marked changes in thebiophysical properties of the RyR2 channel. Such changes are evidencedby increased open probability (Po), due to an increased sensitivity toCa²⁺-dependent activation (Brillantes et al., Stabilization of calciumrelease channel (ryanodine receptor) function by FK506-binding protein.Cell, 77:513-23, 1994; Kaftan et al., Effects of rapamycin on ryanodinereceptor/Ca²⁺-release channels from cardiac muscle. Circ. Res.,78:990-97, 1996); destabilization of the channel, resulting insubconductance states; and impaired coupled gating of the channels,resulting in defective EC coupling and cardiac dysfunction (Marx et al.,Coupled gating between individual skeletal muscle Ca²⁺ release channels(ryanodine receptors). Science, 281:818-21, 1998). Thus,PKA-hyperphosphorylated RyR2 is very sensitive to low-level Ca²⁺stimulation, and this manifests itself as an SR Ca²⁺ leak through thehyperphosphorylated channel.

The maladaptive response to stress in heart failure results in depletionof FKBP12.6 from the channel macromolecular complex. This leads to ashift to the left in the sensitivity of RyR2 to Ca²⁺-induced Ca²⁺release, resulting in channels that are more active at low-to-moderate[Ca²⁺] (Marx et al., PKA phosphorylation dissociates FKBP12.6 from thecalcium release channel (ryanodine receptor): defective regulation infailing hearts. Cell, 101:365-76, 2000; Yamamoto et al., Abnormal Ca²⁺release from cardiac sarcoplasmic reticulum in tachycardia-induced heartfailure. Cardiovasc. Res., 44:146-55, 1999; Yano et al., Alteredstoichiometry of FKBP12.6 versus ryanodine receptor as a cause ofabnormal Ca²⁺ leak through ryanodine receptor in heart failure.Circulation, 102:2131-36, 2000). Over time, the increased “leak” throughRyR2 results in resetting of the SR Ca²⁺ content to a lower level, whichin turn reduces EC coupling gain and contributes to impaired systoliccontractility (Marx et al., PKA phosphorylation dissociates FKBP12.6from the calcium release channel (ryanodine receptor): defectiveregulation in failing hearts. Cell, 101:365-76, 2000).

Additionally, a subpopulation of RyR2 that are particularly “leaky” canrelease SR Ca²⁺ during the resting phase of the cardiac cycle, diastole.This results in depolarizations of the cardiomyocyte membrane known asdelayed after-depolarizations (DADs), which are known to trigger fatalventricular cardiac arrhythmias (Wehrens et al., FKBP12.6 deficiency anddefective calcium release channel (ryanodine receptor) function linkedto exercise-induced sudden cardiac death. Cell, 113:829-40, 2003).

In structurally-normal hearts, a similar phenomenon may be at work.

Specifically, it is known that exercise and stress induce the release ofcatecholamines that activate beta-adrenergic receptors in the heart.Activation of the beta-adrenergic receptors leads tohyperphosphorylation of RyR2 channels. Evidence also suggests that thehyperphosphorylation of RyR2 resulting from beta-adrenergic-receptoractivation renders mutated RyR2 channels more likely to open in therelaxation phase of the cardiac cycle, increasing the likelihood ofarrhythmias.

Cardiac arrhythmias are known to be associated with SR Ca²⁺ leaks instructurally-normal hearts. In these cases, the most common mechanismfor induction and maintenance of ventricular tachycardia is abnormalautomaticity. One form of abnormal automaticity, known as triggeredarrhythmia, is associated with aberrant release of SR Ca²⁺, whichinitiates DADs (Fozzard, H. A., Afterdepolarizations and triggeredactivity. Basic Res. Cardiol., 87:105-13, 1992; Wit and Rosen,Pathophysiologic mechanisms of cardiac arrhythmias. Am. Heart J,106:798-811, 1983). DADs are abnormal depolarizations in cardiomyocytesthat occur after repolarization of a cardiac action potential. Themolecular basis for the abnormal SR Ca²⁺ release that results in DADshas not been fully elucidated. However, DADs are known to be blocked byryanodine, providing evidence that RyR2 may play a key role in thepathogenesis of this aberrant Ca²⁺ release (Marban et al., Mechanisms ofarrhythmogenic delayed and early afterdepolarizations in ferretventricular muscle. J. Clin. Invest., 78:1185-92, 1986; Song andBelardinelli, ATP promotes development of afterdepolarizations andtriggered activity in cardiac myocytes. Am. J. Physiol., 267:H2005-11,1994).

In view of the foregoing, it is clear that leaks in RyR2 channels areassociated with a number of pathological states—in both diseased heartsand structurally-normal hearts. Accordingly, methods to repair the leaksin RyR2 could prevent heart failure, and fatal arrhythmias andfibrillations, in millions of patients.

JTV-519(4-[3-(4-benzylpiperidin-1-yl)propionyl]-7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepinemonohydrochloride; also known as k201 or ICP-Calstan 100), a derivativeof 1,4-benzothiazepine, is a new modulator of calcium-ion channels. Inaddition to regulating Ca²⁺ levels in myocardial cells, JTV-519 alsomodulates the Na⁺ current and the inward-rectifier K⁺ current in guineapig ventricular cells, and inhibits the delayed-rectifier K⁺ current inguinea pig atrial cells. Studies have shown that JTV-519 has a strongcardioprotective effect against catecholamine-induced myocardial injury,myocardial-injury-induced myofibrillar overcontraction, andischemia/reperfusion injury. In experimental myofibrillarovercontraction models, JTV-519 demonstrated greater cardioprotectiveeffects than propranolol, verapamil, and diltiazem. Experimental dataalso suggest that JTV-519 effectively prevents ventricularischemia/reperfusion by reducing the level of intracellular Ca²⁺overload in animal models.

SUMMARY OF THE INVENTION

The present invention is based upon the surprising discovery that RyR2is a target for treating and preventing heart failure and cardiacarrhythmias, including atrial fibrillations and cardiac arrhythmias thatcause exercise-induced sudden cardiac death (SCD).

As described herein, the inventors made mutant RyR2 channels with 7different CPVT mutations, and studied their functions. All 7 mutants hadfunctional defects that resulted in channels that became leaky (acalcium leak) when stimulated during exercise. The inventors' study isthe first to identify a mechanism by which the SR calcium leak causesDADs. Remarkably, the defect in the mutant CPVT channels made thechannels look like the leaky channels in the hearts of patients withend-stage heart failure—a disorder characterized by a high incidence offatal cardiac arrhythmias. Therefore, the inventors demonstrate hereinthat the mechanism for the VT in CPVT is the same as the mechanism forVT in heart failure.

The inventors also disclose herein that the drug JTV-519 (k201 orICP-Calstan 100), a member of the 1,4 benzothiazepine family ofcompounds, repairs the leak in RyR2 channels. As the inventors showherein, JTV-519 enhances binding of FKBP12.6 to PKA-phosphorylated RyR2,and to mutant RyR2s that otherwise have reduced affinity for, or do notbind to, FKBP12.6. This action of JTV-519 fixes the leak in RyR2 thattriggers fatal cardiac arrhythmias (cardiac death) and that contributesto atrial/ventricular fibrillations and heart muscle dysfunction inheart failure.

Accordingly, in one aspect, the present invention provides novel1,4-benzothiazepine intermediates and derivatives, as well as methodsfor synthesizing same, and methods for assaying for same. In certainembodiments, these novel 1,4-benzothiazepine intermediates andderivatives include:

wherein R=aryl, alkenyl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, andn=0, 1, 2, or 3, and R′=alkyl or cycloalkyl;

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, and n=0, 1, 2,or 3, and R′=alkyl or cycloalkyl;

wherein R=CO(CH₂)_(n)XR′₂, SO₂(CH₂)_(n)XR′₂, or SO₂NH(CH₂)_(n)XR′₂, andX=N or S, and n=1, 2, or 3, and R′=alkyl or cycloalkyl; and wherein m=1or 2;

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, —(CH₂)_(n)SR′, and n=0, 1, 2, or3, and R′=alkyl or cycloalkyl; and wherein X=NH or O; and

wherein R₁=OR′, SR′, NR′, alkyl, or halide, at position 2, 3, 4, or 5 onthe phenyl ring, and R′=alkyl, aryl, or H; wherein R₂=H, alkyl, or aryl;and wherein R₃=H, alkyl, or aryl.

Additional embodiments may include the following compounds: S7, S-20,S-25, S-27, and S36 (set forth in Appendix A).

Also provided are uses of the novel 1,4-benzothiazepine compounds inmethods for limiting or preventing a decrease in the level of RyR2-boundFKBP12.6 in a subject; in methods for treating or preventing heartfailure, atrial fibrillation, or exercise-induced cardiac arrhythmia ina subject; and in methods for preventing exercise-induced sudden cardiacdeath in a subject.

In a further aspect, the present invention provides a method foridentifying an agent that enhances binding of RyR2 and FKBP12.6,comprising the steps of: (a) obtaining or generating a source of RyR2;(b) exposing the RyR2 to FKBP12.6, in the presence of a candidate agent;and (c) determining if the agent enhances the binding of RyR2 andFKBP12.6. In certain embodiments, the RyR2 may be unphosphorylated,PKA-phosphorylated, or PKA-hyperphosphorylated. Also provided are anagent identified by this method, and uses of the agent in methods forlimiting or preventing a decrease in the level of RyR2-bound FKBP12.6 ina subject; in methods for treating or preventing heart failure, atrialfibrillation, or exercise-induced cardiac arrhythmia in a subject; andin methods for preventing exercise-induced sudden cardiac death in asubject.

In still another aspect, the present invention provides a method foridentifying an agent for enhancing the binding of RyR2 and FKBP12.6,comprising the steps of: (a) obtaining or generating a source ofFKBP12.6; (b) exposing the FKBP12.6 to RyR2, in the presence of acandidate agent; and (c) determining if the agent enhances the bindingof RyR2 and FKBP12.6. In certain embodiments, the RyR2 may beunphosphorylated, PKA-phosphorylated, or PKA-hyperphosphorylated. Alsoprovided are an agent identified by this method, and uses of the agentin methods for limiting or preventing a decrease in the level ofRyR2-bound FKBP12.6 in a subject; in methods for treating or preventingheart failure, atrial fibrillation, or exercise-induced cardiacarrhythmia in a subject; and in methods for preventing exercise-inducedsudden cardiac death in a subject.

Additional aspects of the present invention will be apparent in view ofthe description which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates that JTV-519 prevents exercise-induced ventriculararrhythmias in FKBP12.6^(+/−) mice. (A) Representative ambulatoryelectrocardiograms of an untreated FKBP12.6^(+/−) mouse, anFKBP12.6^(+/−) mouse treated with JTV-519, and an FKBP12.6^(−/−) mousetreated with JTV-519. There were no significant differences in heartrate, or in any of the measured ECG parameters. (B) upper tracing:Example of sustained polymorphic ventricular tachycardia, recorded in anuntreated FKBP12.6^(+/−) mouse subjected to exercise testing andinjection with 1.0 mg/kg epinephrine. middle tracing: Electro-cardiogramof a JTV-519-treated FKBP12.6^(+/−) mouse following the same protocol;no arrhythmias were detected. bottom tracing: Exercise-inducedventricular tachycardia (VT) in an FKBP12.6^(−/−) mouse treated withJTV-519. The dotted line represents 16.31 seconds of VT that are notshown in the figure. ‘P’ indicates a P-wave, which is indicative ofsinus rhythm following ventricular tachycardia. (C) Bar graph showingquantification of sudden cardiac death (left), sustained ventriculartachycardias (>10 beats, middle), and non-sustained ventriculartachycardias (3-10 abnormal beats, right) in FKBP12.6^(+/−) andFKBP12.6^(−/−) mice, either treated or not treated with JTV-519,respectively. It should be noted that treatment with JTV-519 completelyprevented exercise- and epinephrine-induced arrhythmias inFKBP12.6^(+/−) mice treated with JTV-519 (n=9), as compared withuntreated FKBP12.6^(+/−) mice (n=10) or JTV-519-treated FKBP12.6^(−/−)mice (n=5), suggesting that JTV-519 prevents arrhythmias and suddendeath in FKBP12.6^(+/−) mice by rebinding FKBP12.6 to RyR2.

FIG. 2 shows that JTV-519 prevents exercise-induced sudden cardiac death(SCD) by increasing the affinity of FKBP12.6 for RyR2 in FKBP12.6^(+/−)mice. (A-B) Cardiac ryanodine receptors (RyR2) were immunoprecipitatedusing RyR2-5029 antibody. Shown are immunoblots (A) and bar graphs (B)representing the quantified amounts of RyR2, PKA-phosphorylated RyR2(RyR2-pSer²⁸⁰⁹ antibody), and FKBP12.6 in wild-type (FKBP12.6^(+/+))mice, FKBP12.6^(+/−) mice, and FKBP12.6^(−/−) under resting conditions,and following exercise, either in the absence or presence of JTV-519,respectively. Under resting conditions, 70% of FKBP12.6 is associatedwith RyR2 in FKBP12.6^(+/−) mice. Following exercise testing, the amountof FKBP12.6 associated with the RyR2 complex was dramatically decreasedin FKBP12.6^(+/−) mice, but this could be rescued by treatment withJTV-519. (C) RyR2 single channels were isolated from hearts obtainedfollowing exercise testing and epinephrine injection. Shown are channelsfrom FKBP12.6^(+/−) mice, with and without pre-treatment with JTV-519,and channels from FKBP12.6^(−/−) mice following JTV-519 pre-treatment.It should be noted that RyR2-channel function was normalized in theexercised FKBP12.6^(+/−) mouse treated with JTV-519. The representativesingle channel from an exercised FKBP12.6^(−/−) mouse after JTV-519treatment shows that FKBP12.6 in the heart is required for the action ofJTV-519. The dotted lines represent incomplete channel openings, or‘subconductance’ openings, and are indicative of FKBP12.6-depleted RyR2channels. Tracings on the left represent 5.0 sec, while tracings on theright represent 500 msec. In the figure, Po=open probability; To=averageopen times; Tc=average closed times; and c=closed state of the channel.(D) Summary bar graph showing average open probabilities of single RyR2channels (see above). JTV-519 dramatically reduces the open probabilityof RyR2 from FKBP12.6^(+/−) mice following exercise testing at diastoliccalcium concentrations (150 nM).

FIG. 3 illustrates JTV-519 normalizes RyR2-channel gating by increasedFKBP12.6 binding affinity to PKA-phosphorylated RyR2 channels. (A, B)Canine cardiac SR membranes (A) and recombinantly-expressed RyR2channels (B) were prepared as described previously (Kaftan et al.,Effects of rapamycin on ryanodine receptor/Ca⁽²⁺⁾-release channels fromcardiac muscle. Circ. Res., 78:990-97, 1996). (A) Ryanodine receptors(RyR2) were phosphorylated with PKA catalytic subunit (40 U; SigmaChemical Co., St. Louis, Mo.), in the presence or absence of the PKAinhibitor, PKI₅₋₂₄, in phosphorylation buffer (8 mM MgCl₂, 10 mM EGTA,and 50 mM Tris/PIPES; pH 6.8). Samples were centrifuged at 100,000×g for10 min, and washed three times in imidazole buffer (10 mM imidazole; pH7). Recombinantly-expressed FKBP12.6 (final concentration=250 nM) wasadded to the samples, in the absence or presence of differentconcentrations of JTV-519. After a 60-min incubation, samples werecentrifuged at 100,000×g for 10 min, and washed twice in imidazolebuffer. Samples were heated to 95° C., and size-fractionated usingSDS-PAGE. Immunoblotting of the SR microsomes was performed, aspreviously described (Jayaraman et al., FK506 binding protein associatedwith the calcium release channel (ryanodine receptor). J. Biol. Chem.,267:9474-77, 1992), with anti-FKBP12.6 antibody (1:1,000) andanti-RyR2-5029 antibody (1:3,000). The figure demonstrates that JTV-519enables FKBP12.6 to bind to: (A) PKA-phosphorylated RyR2 (partialbinding at 100 nM; complete binding at 1000 nM) or (B) RyR2-S2809Dmutant channels, which are constitutively PKA-phosphorylated RyR2channels. (C-E) Single-channel studies showing increased openprobability of RyR2 following PKA phosphorylation (D), as compared withPKA phosphorylation in the presence of the specific PKA inhibitor,PKI₅₋₂₄ (C). Single-channel function was normalized inPKA-phosphorylated RyR2 incubated with FKBP12.6 in the presence ofJTV-519 (E). Channel openings are upward, the dash indicates the levelof full openings (4 pA), and the letter ‘c’ indicates the closed state.Channels are shown at compressed (5 sec, upper tracing) and expanded(500 msec, lower tracing) time scales, and recordings are at 0 mV.Amplitude histograms (right) revealed increased activity andsubconductance openings in PKA-phosphorylated RyR2, but not followingtreatment with JTV-519 and FKBP12.6. (F) Normalized plot of openprobability as a function of cytosolic [Ca²⁺]. Incubation ofPKA-phosphorylated RyR2 with FKBP12.6 in the presence of JTV-519 shiftedthe Ca²⁺-dependence of RyR2 activation towards the right, making itsimilar to the Ca²⁺-dependence of unphosphorylated channels.

FIG. 4 demonstrates the experimental protocol used to test effects ofthe inventors' novel JTV-519-related compounds (disclosed herein) onhERG-channel current. Whole-cell patch-clamp experiments were carriedout with physiological solutions at room temperature, in CHO cellstransfected with hERG channel. Voltage-clamp protocols are shown in thelower panels. In vehicle, 0.1% DMSO in the external solution was appliedwith the same time-protocol as that shown in the upper panel.

FIG. 5 illustrates the effects of JTV-519 and the inventors' novelJTV-519-related compound, S36 (disclosed herein), on hERG-channelcurrents elicited by 80-mV depolarization. Representative hERG-channelcurrents (I(Kr)) were recorded from CHO cells before (open circle) andafter (closed circle) application of 1 μM JTV-519 (left panel) or 1 μMJTV-S36 (right panel). The voltage-clamp protocol is shown below thecurrent traces. Currents were elicited during 400-msec depolarization to+80 mV, from a holding potential of −90 mV. It should be noted that,upon the 400-msec depolarization (which mimics the human actionpotential duration (APD)), hERG channels pass very little outwardcurrent, because they rapidly inactivate. Tail currents marked bycircles in current traces were elicited by return of the membranepotential to −40 mV, in the recovery from inactivation through the openstate. Because the tail current is a major contributor to control of theAPD, effects of the drugs were evaluated by tail currents at −40 mV:JTV-519=83% block; JTV-S36=39% block.

FIG. 6 shows effects of JTV-519, E4031, and the inventors' novelJTV-519-related compound, S36 (disclosed herein), on activation ofhERG-channel currents (traces). Representative hERG-channel I-Vrelationships are shown before (control, left panels) and after (centralpanels) application of 0.1% DMSO (vehicle; upper central panel), 1 μMJTV-519 (middle central panel), and 1 μM JTV-S36 (lower central panel).The right panel shows that 5 μM E4031 (a class III anti-arrhythmic drugknown to block hERG channels) completely blocked hERG-channel currents.(Note the tail currents at −40 mV). The voltage-clamp protocol is setforth in FIG. 4, as an I-V relationship.

FIG. 7 demonstrates effects of JTV-519 and the inventors' novelJTV-519-related compound, S36 (disclosed herein), on activation ofhERG-channel currents. The hERG-channel I-V relationships are shown forpeak tail currents (activation) before (open squares) and after (closedsquares) application of 0.1% DMSO (vehicle; upper panel, 1 μM JTV-519(lower left panel), and 1 μM JTV-S36 (lower right panel). Washout of thedrugs is depicted with open triangles. The voltage-clamp protocol is setforth in FIG. 4, as an I-V relationship. It should be noted that JTV-S36did not block hERG currents at negative potentials (0 mV; 20 mVdepolarization) showing voltage-dependent block of I(Kr).

FIG. 8 shows the structures of the derivatives.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, catecholaminergic polymorphic ventriculartachycardia (CPVT) is an inherited disorder in individuals withstructurally-normal hearts. It is characterized by stress-inducedventricular tachycardia, a lethal arrhythmia that may cause suddencardiac death (SCD). Mutations in RyR2 channels, located on thesarcoplasmic reticulum (SR), have been linked to CPVT. To determine themolecular mechanism underlying the fatal cardiac arrhythmias in CPVT,the inventors studied CPVT-associated mutant RyR2 channels (e.g.,S2246L, R2474S, N4104K, R4497C).

All individuals with CPVT have exercise-induced cardiac arrhythmias. Theinventors previously showed that exercise-induced arrhythmias and suddendeath (in patients with CPVT) result from a reduced affinity of FKBP12.6for RyR2. Herein, the inventors have demonstrated that exerciseactivates RyR2 as a result of phosphorylation by adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase (PKA). Mutant RyR2channels, which had normal function in planar lipid bilayers under basalconditions, were more sensitive to activation by PKAphosphorylation—exhibiting increased activity (open probability) andprolonged open states, as compared with wild-type channels. In addition,PKA-phosphorylated mutant RyR2 channels were resistant to inhibition byMg²⁺, a physiological inhibitor of the channel, and showed reducedbinding to FKBP12.6 (which stabilizes the channel in the closed state).These findings indicate that, during exercise, when the RyR2 arePKA-phosphorylated, the mutant CPVT channels are more likely to open inthe relaxation phase of the cardiac cycle (diastole), increasing thelikelihood of arrhythmias triggered by SR Ca²⁺ leak. Since heart failureis a leading cause of death worldwide, methods to repair the leak inRyR2 could prevent fatal arrhythmias in millions of patients.

The inventors have further demonstrated herein that JTV-519, abenzothiazepine derivative, prevents lethal ventricular arrhythmias inmice heterozygous for the FKBP12.6 gene. JTV-519 reduced the openprobability of RyR2, isolated from FKBP12.6^(+/−) mice that diedfollowing exercise, by increasing the affinity of FKBP12.6 forPKA-phosphorylated RyR2. Moreover, JTV-519 normalized gating ofCPVT-associated mutant RyR2 channels by increasing FKBP12.6 bindingaffinity. These data indicate that JTV-519 may prevent fatal ventriculararrhythmias by increasing FKBP12.6-RyR2 binding affinity.

Novel Methods of Treatment and Prevention

In accordance with the foregoing, the present invention provides amethod for limiting or preventing a decrease in the level of RyR2-boundFKBP12.6 in cells of a subject.

As used herein, “FKBP12.6” includes both an “FKBP12.6 protein” and an“FKBP12.6 analogue”. Unless otherwise indicated herein, “protein” shallinclude a protein, protein domain, polypeptide, or peptide, and anyfragment thereof. An “FKBP12.6 analogue” is a functional variant of theFKBP12.6 protein, having FKBP12.6 biological activity, that has 60% orgreater amino-acid-sequence homology with the FKBP12.6 protein. Asfurther used herein, the term “FKBP12.6 biological activity” refers tothe activity of a protein or peptide that demonstrates an ability toassociate physically with, or bind with, unphosphorylated ornon-hyperphosphorylated RyR2 (i.e., binding of approximately two fold,or, more preferably, approximately five fold, above the backgroundbinding of a negative control), under the conditions of the assaysdescribed herein, although affinity may be different from that ofFKBP12.6.

In addition, as used herein, “RyR2” includes both an “RyR2 protein” andan “RyR2 analogue”. An “RyR2 analogue” is a functional variant of theRyR2 protein, having RyR2 biological activity, that has 60% or greateramino-acid-sequence homology with the RyR2 protein. As used herein, theterm “RyR2 analogue” includes RyR1—the skeletal-muscle isoform of RyR2.The RyR2 of the present invention may be unphosphorylated,phosphorylated (e.g., by PKA), or hyperphosphorylated (e.g., by PKA). Asfurther used herein, the term “RyR2 biological activity” refers to theactivity of a protein or peptide that demonstrates an ability toassociate physically with, or bind with, FKBP12.6 (i.e., binding ofapproximately two fold, or, more preferably, approximately five fold,above the background binding of a negative control), under theconditions of the assays described herein, although affinity may bedifferent from that of RyR2.

As described above, the cardiac ryanodine receptor, RyR2, is a proteincomplex comprising four 565,000-dalton RyR2 proteins in association withfour 12,000-dalton FKBP12.6 proteins. FK506 binding proteins (FKBPs) arecis-trans peptidyl-prolyl isomerases that are widely expressed, andserve a variety of cellular functions. FKBP12.6 protein is tightly boundto, and regulates the function of, RyR2. FKBP12.6 binds to the RyR2channel, one molecule per RyR2 subunit, stabilizes RyR2-channelfunction, and facilitates coupled gating between neighboring RyR2channels, thereby preventing aberrant activation of the channel duringthe resting phase of the cardiac cycle. Accordingly, as used herein, theterm “RyR2-bound FKBP12.6” includes a molecule of an FKBP12.6 proteinthat is bound to an RyR2 protein subunit or a tetramer of FKBP12.6 thatis in association with a tetramer of RyR2.

In accordance with the method of the present invention, a “decrease” inthe level of RyR2-bound FKBP12.6 in cells of a subject refers to adetectable decrease, diminution, or reduction in the level of RyR2-boundFKBP12.6 in cells of the subject. Such a decrease is limited orprevented in cells of a subject when the decrease is in any way halted,hindered, impeded, obstructed, or reduced by the administration ofJTV-519 (as described below), such that the level of RyR2-bound FKBP12.6in cells of the subject is higher than it would otherwise be in theabsence of JTV-519.

The level of RyR2-bound FKBP12.6 in a subject may be detected bystandard assays and techniques, including those readily determined fromthe known art (e.g., immunological techniques, hybridization analysis,immunoprecipitation, Western-blot analysis, fluorescence imagingtechniques, and/or radiation detection, etc.), as well as any assays anddetection methods disclosed herein. For example, protein may be isolatedand purified from cells of a subject using standard methods known in theart, including, without limitation, extraction from the cells (e.g.,with a detergent that solubilizes the protein) where necessary, followedby affinity purification on a column, chromatography (e.g., FTLC andHPLC), immunoprecipitation (with an antibody), and precipitation (e.g.,with isopropanol and a reagent such as Trizol). Isolation andpurification of the protein may be followed by electrophoresis (e.g., onan SDS-polyacrylamide gel). A decrease in the level of RyR2-boundFKBP12.6 in a subject, or the limiting or prevention thereof, may bedetermined by comparing the amount of RyR2-bound FKBP12.6 detected priorto the administration of JTV-519 (in accordance with methods describedbelow) with the amount detected a suitable time after administration ofJTV-519.

In the method of the present invention, a decrease in the level ofRyR2-bound FKBP12.6 in cells of a subject may be limited or prevented,for example, by inhibiting dissociation of FKBP12.6 and RyR2 in cells ofthe subject; by increasing binding between FKBP12.6 and RyR2 in cells ofthe subject; or by stabilizing the RyR2-FKBP12.6 complex in cells of asubject. As used herein, the term “inhibiting dissociation” includesblocking, decreasing, inhibiting, limiting, or preventing the physicaldissociation or separation of an FKBP12.6 subunit from an RyR2 moleculein cells of the subject, and blocking, decreasing, inhibiting, limiting,or preventing the physical dissociation or separation of an RyR2molecule from an FKBP12.6 subunit in cells of the subject. As furtherused herein, the term “increasing binding” includes enhancing,increasing, or improving the ability of phosphorylated RyR2 to associatephysically with FKBP12.6 (e.g., binding of approximately two fold, or,more preferably, approximately five fold, above the background bindingof a negative control) in cells of the subject, and enhancing,increasing, or improving the ability of FKBP12.6 to associate physicallywith phosphorylated RyR2 (e.g., binding of approximately two fold, or,more preferably, approximately five fold, above the background bindingof a negative control) in cells of the subject. Additionally, in themethod of the present invention, a decrease in the level of RyR2-boundFKBP12.6 in cells of a subject may be limited or prevented by directlydecreasing the level of phosphorylated RyR2 in cells of the subject, orby indirectly decreasing the level of phosphorylated RyR2 in the cells(e.g., by targeting an enzyme (such as PKA) or another endogenousmolecule that regulates or modulates the functions or levels ofphosphorylated RyR2 in the cells). Preferably, the level ofphosphorylated RyR2 in the cells is decreased by at least 10% in themethod of the present invention. More preferably, the level ofphosphorylated RyR2 is decreased by at least 20%.

In accordance with the method of the present invention, a decrease inthe level of RyR2-bound FKBP12.6 is limited or prevented in a subject,particularly in cells of a subject. The subject of the present inventionmay be any animal, including amphibians, birds, fish, mammals, andmarsupials, but is preferably a mammal (e.g., a human; a domesticanimal, such as a cat, dog, monkey, mouse, or rat; or a commercialanimal, such as a cow or pig). Additionally, the subject of the presentinvention is a candidate for exercise-induced cardiac arrhythmia.Exercise-induced cardiac arrhythmia is a heart condition (e.g., aventricular fibrillation or ventricular tachycardia, including any thatleads to sudden cardiac death) that develops during/after a subject hasundergone physical exercise. A “candidate” for exercise-induced cardiacarrhythmia is a subject who is known to be, or is believed to be, or issuspected of being, at risk for developing cardiac arrhythmiaduring/after physical exercise. Examples of candidates forexercise-induced cardiac arrhythmia include, without limitation, ananimal/person known to have catecholaminergic polymorphic ventriculartachycardia (CPVT); an animal/person suspected of having CPVT; and ananimal/person who is known to be, or is believed to be, or is suspectedof being, at risk for developing cardiac arrhythmia during/afterphysical exercise, and who is about to exercise, is currentlyexercising, or has just completed exercise. As discussed above, CPVT isan inherited disorder in individuals with structurally-normal hearts. Itis characterized by stress-induced ventricular tachycardia—a lethalarrhythmia that may cause sudden cardiac death. In subjects with CPVT,physical exertion and/or stress induce bidirectional and/or polymorphicventricular tachycardias that lead to sudden cardiac death (SCD) in theabsence of detectable structural heart disease. Individuals with CPVThave ventricular arrhythmias when subjected to exercise, but do notdevelop arrhythmias at rest.

In the method of the present invention, the cells of a subject arepreferably striated muscle cells. A striated muscle is a muscle in whichthe repeating units (sarcomeres) of the contractile myofibrils arearranged in registry throughout the cell, resulting in transverse oroblique striations that may be observed at the level of a lightmicroscope. Examples of striated muscle cells include, withoutlimitation, voluntary (skeletal) muscle cells and cardiac muscle cells.In a preferred embodiment, the cell used in the method of the presentinvention is a human cardiac muscle cell. As used herein, the term“cardiac muscle cell” includes cardiac muscle fibers, such as thosefound in the myocardium of the heart. Cardiac muscle fibers are composedof chains of contiguous heart-muscle cells, or cardiomyocytes, joinedend to end at intercalated disks. These disks possess two kinds of celljunctions: expanded desmosomes extending along their transverseportions, and gap junctions, the largest of which lie along theirlongitudinal portions.

In the method of the present invention, a decrease in the level ofRyR2-bound FKBP12.6 is limited or prevented in cells of a subject byadministering JTV-519 to the subject; this would also permit contactbetween cells of the subject and JTV-519. JTV-519(4-[3-(4-benzylpiperidin-1-yl)propionyl]-7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepinemonohydrochloride), also known as k201, is a derivative of1,4-benzothiazepine, and a modulator of calcium-ion channels. Inaddition to regulating Ca²⁺ levels in myocardial cells, JTV-519modulates the Na⁺ current and the inward-rectifier K⁺ current in guineapig ventricular cells, and inhibits the delayed-rectifier K⁺ current inguinea pig atrial cells. FK506 and rapamycin are drugs that may be usedto design other compounds that stabilize RyR2-FKBP12.6 binding in cellsof a subject who is a candidate for exercise-induced cardiac arrhythmia.FK506 and rapamycin both dissociate FKBP12.6 from RyR2. It is possibleto design and/or screen for compounds that are structurally related tothese drugs, but have the opposite effects.

In the method of the present invention, JTV-519 may be administered to asubject by way of a therapeutic composition, comprising JTV-519 and apharmaceutically-acceptable carrier. The pharmaceutically-acceptablecarrier must be “acceptable” in the sense of being compatible with theother ingredients of the composition, and not deleterious to therecipient thereof. The pharmaceutically-acceptable carrier employedherein is selected from various organic or inorganic materials that areused as materials for pharmaceutical formulations, and which may beincorporated as analgesic agents, buffers, binders, disintegrants,diluents, emulsifiers, excipients, extenders, glidants, solubilizers,stabilizers, suspending agents, tonicity agents, vehicles, andviscosity-increasing agents. If necessary, pharmaceutical additives,such as antioxidants, aromatics, colorants, flavor-improving agents,preservatives, and sweeteners, may also be added. Examples of acceptablepharmaceutical carriers include carboxymethyl cellulose, crystallinecellulose, glycerin, gum arabic, lactose, magnesium stearate, methylcellulose, powders, saline, sodium alginate, sucrose, starch, talc, andwater, among others.

The pharmaceutical formulations of the present invention may be preparedby methods well-known in the pharmaceutical arts. For example, theJTV-519 may be brought into association with a carrier or diluent, as asuspension or solution. Optionally, one or more accessory ingredients(e.g., buffers, flavoring agents, surface active agents, and the like)also may be added. The choice of carrier will depend upon the route ofadministration.

JTV-519 may be administered to a subject by contacting target cells(e.g., cardiac muscle cells) in vivo in the subject with the JTV-519.JTV-519 may be contacted with (e.g., introduced into) cells of thesubject using known techniques utilized for the introduction andadministration of proteins, nucleic acids, and other drugs. Examples ofmethods for contacting the cells with (i.e., treating the cells with)JTV-519 include, without limitation, absorption, electroporation,immersion, injection, introduction, liposome delivery, transfection,transfusion, vectors, and other drug-delivery vehicles and methods. Whenthe target cells are localized to a particular portion of a subject, itmay be desirable to introduce the JTV-519 directly to the cells, byinjection or by some other means (e.g., by introducing the JTV-519 intothe blood or another body fluid). The target cells may be contained inheart tissue of a subject, and may be detected in heart tissue of thesubject by standard detection methods readily determined from the knownart, examples of which include, without limitation, immunologicaltechniques (e.g., immunohistochemical staining), fluorescence imagingtechniques, and microscopic techniques.

Additionally, the JTV-519 of the present invention may be administeredto a human or animal subject by known procedures, including, withoutlimitation, oral administration, parenteral administration, andtransdermal administration. Preferably, the JTV-519 is administeredparenterally, by epifascial, intracapsular, intracranial,intracutaneous, intrathecal, intramuscular, intraorbital,intraperitoneal, intraspinal, intrasternal, intravascular, intravenous,parenchymatous, subcutaneous, or sublingual injection, or by way ofcatheter. In one embodiment, the agent is administered to the subject byway of targeted delivery to cardiac muscle cells via a catheter insertedinto the subject's heart.

For oral administration, a JTV-519 formulation may be presented ascapsules, tablets, powders, granules, or as a suspension. Theformulation may have conventional additives, such as lactose, mannitol,corn starch, or potato starch. The formulation also may be presentedwith binders, such as crystalline cellulose, cellulose derivatives,acacia, corn starch, or gelatins. Additionally, the formulation may bepresented with disintegrators, such as corn starch, potato starch, orsodium carboxymethylcellulose. The formulation also may be presentedwith dibasic calcium phosphate anhydrous or sodium starch glycolate.Finally, the formulation may be presented with lubricants, such as talcor magnesium stearate.

For parenteral administration (i.e., administration by injection througha route other than the alimentary canal), JTV-519 may be combined with asterile aqueous solution that is preferably isotonic with the blood ofthe subject. Such a formulation may be prepared by dissolving a solidactive ingredient in water containing physiologically-compatiblesubstances, such as sodium chloride, glycine, and the like, and having abuffered pH compatible with physiological conditions, so as to producean aqueous solution, then rendering said solution sterile. Theformulation may be presented in unit or multi-dose containers, such assealed ampoules or vials. The formulation may be delivered by any modeof injection, including, without limitation, epifascial, intracapsular,intracranial, intracutaneous, intrathecal, intramuscular, intraorbital,intraperitoneal, intraspinal, intrasternal, intravascular, intravenous,parenchymatous, subcutaneous, or sublingual, or by way of catheter intothe subject's heart.

For transdermal administration, JTV-519 may be combined with skinpenetration enhancers, such as propylene glycol, polyethylene glycol,isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and the like,which increase the permeability of the skin to the JTV-519, and permitthe JTV-519 to penetrate through the skin and into the bloodstream. TheJTV-519/enhancer composition also may be further combined with apolymeric substance, such as ethylcellulose, hydroxypropyl cellulose,ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to providethe composition in gel form, which may be dissolved in a solvent, suchas methylene chloride, evaporated to the desired viscosity, and thenapplied to backing material to provide a patch.

In accordance with the method of the present invention, JTV-519 may beadministered to the subject (and JTV-519 may be contacted with cells ofthe subject) in an amount effective to limit or prevent a decrease inthe level of RyR2-bound FKBP12.6 in the subject, particularly in cellsof the subject. This amount may be readily determined by the skilledartisan, based upon known procedures, including analysis of titrationcurves established in vivo, and methods and assays disclosed herein. Asuitable amount of JTV-519 effective to limit or prevent a decrease inthe level of RyR2-bound FKBP12.6 in the subject may range from about 5mg/kg/day to about 20 mg/kg/day, and/or may be an amount sufficient toachieve plasma levels ranging from about 300 ng/ml to about 1000 ng/ml.Preferably, the amount of JTV-519 ranges from about 10 mg/kg/day toabout 20 mg/kg/day.

In one embodiment of the present invention, the subject has not yetdeveloped exercise-induced cardiac arrhythmia. In this case, the amountof JTV-519 effective to limit or prevent a decrease in the level ofRyR2-bound FKBP12.6 in the subject may be an amount of JTV-519 effectiveto prevent exercise-induced cardiac arrhythmia in the subject. Cardiacarrhythmia is a disturbance of the electrical activity of the heart thatmanifests as an abnormality in heart rate or heart rhythm. As usedherein, an amount of JTV-519 “effective to prevent exercise-inducedcardiac arrhythmia” includes an amount of JTV-519 effective to preventthe development of the clinical impairment or symptoms of theexercise-induced cardiac arrhythmia (e.g., palpitations, fainting,ventricular fibrillation, ventricular tachycardia, and sudden cardiacdeath). The amount of JTV-519 effective to prevent exercise-inducedcardiac arrhythmia in a subject will vary depending upon the particularfactors of each case, including the type of exercise-induced cardiacarrhythmia, the subject's weight, the severity of the subject'scondition, and the mode of administration of the JTV-519. This amountmay be readily determined by the skilled artisan, based upon knownprocedures, including clinical trials, and methods disclosed herein. Ina preferred embodiment, the amount of JTV-519 effective to prevent theexercise-induced cardiac arrhythmia is an amount of JTV-519 effective toprevent exercise-induced sudden cardiac death in the subject. In anotherpreferred embodiment, the JTV-519 prevents exercise-induced cardiacarrhythmia and exercise-induced sudden cardiac death in the subject.

Because of its ability to stabilize RyR2-bound FKBP12.6, and maintainand restore balance in the context of dynamic PKA phosphorylation anddephosphorylation of RyR2, JTV-519 may also be useful in treating asubject who has already started to experience clinical symptoms ofexercise-induced cardiac arrhythmia. If the symptoms of arrhythmia areobserved in the subject early enough, JTV-519 might be effective inlimiting or preventing a further decrease in the level of RyR2-boundFKBP12.6 in the subject.

Accordingly, in still another embodiment of the present invention, thesubject has been exercising, or is currently exercising, and hasdeveloped exercise-induced cardiac arrhythmia. In this case, the amountof JTV-519 effective to limit or prevent a decrease in the level ofRyR2-bound FKBP12.6 in the subject may be an amount of JTV-519 effectiveto treat exercise-induced cardiac arrhythmia in the subject. As usedherein, an amount of JTV-519 “effective to treat exercise-inducedcardiac arrhythmia” includes an amount of JTV-519 effective to alleviateor ameliorate the clinical impairment or symptoms of theexercise-induced cardiac arrhythmia (e.g., palpitations, fainting,ventricular fibrillation, ventricular tachycardia, and sudden cardiacdeath). The amount of JTV-519 effective to treat exercise-inducedcardiac arrhythmia in a subject will vary depending upon the particularfactors of each case, including the type of exercise-induced cardiacarrhythmia, the subject's weight, the severity of the subject'scondition, and the mode of administration of the JTV-519. This amountmay be readily determined by the skilled artisan, based upon knownprocedures, including clinical trials, and methods disclosed herein. Ina preferred embodiment, the JTV-519 treats exercise-induced cardiacarrhythmia in the subject.

The present invention further provides a method for treatingexercise-induced cardiac arrhythmia in a subject. The method comprisesadministering JTV-519 to the subject in an amount effective to treatexercise-induced cardiac arrhythmia in the subject. A suitable amount ofJTV-519 effective to treat exercise-induced cardiac arrhythmia in thesubject may range from about 5 mg/kg/day to about 20 mg/kg/day, and/ormay be an amount sufficient to achieve plasma levels ranging from about300 ng/ml to about 1000 ng/ml. The present invention also provides amethod for preventing exercise-induced cardiac arrhythmia in a subject.The method comprises administering JTV-519 to the subject in an amounteffective to prevent exercise-induced cardiac arrhythmia in the subject.A suitable amount of JTV-519 effective to prevent exercise-inducedcardiac arrhythmia in the subject may range from about 5 mg/kg/day toabout 20 mg/kg/day, and/or may be an amount sufficient to achieve plasmalevels ranging from about 300 ng/ml to about 1000 ng/ml. Additionally,the present invention provides a method for preventing exercise-inducedsudden cardiac death in a subject. The method comprises administeringJTV-519 to the subject in an amount effective to preventexercise-induced sudden cardiac death in the subject. A suitable amountof JTV-519 effective to prevent exercise-induced sudden cardiac death inthe subject may range from about 5 mg/kg/day to about 20 mg/kg/day,and/or may be an amount sufficient to achieve plasma levels ranging fromabout 300 ng/ml to about 1000 ng/ml.

In various embodiments of the above-described methods, theexercise-induced cardiac arrhythmia in the subject is associated withVT. In preferred embodiments, the VT is CPVT. In other embodiments ofthese methods, the subject is a candidate for exercise-induced cardiacarrhythmia, including candidates for exercise-induced sudden cardiacdeath.

In view of the foregoing methods, the present invention also providesuse of JTV-519 in a method for limiting or preventing a decrease in thelevel of RyR2-bound FKBP12.6 in a subject who is a candidate forexercise-induced cardiac arrhythmia. The present invention also providesuse of JTV-519 in a method for treating or preventing exercise-inducedcardiac arrhythmia in a subject. Furthermore, the present inventionprovides use of JTV-519 in a method for preventing exercise-inducedsudden cardiac death in a subject.

As discussed above and presented herein, the inventors' data show thatprotein kinase A (PKA) phosphorylation of the cardiac ryanodinereceptor, RyR2, on serine 2809 activates the channel by releasing theFK506 binding protein, FKBP12.6. In failing hearts (including humanhearts and animal models of heart failure), RyR2 isPKA-hyperphosphorylated, resulting in defective channels that havedecreased amounts of bound FKBP12.6, and have increased sensitivity tocalcium-induced activation. The net result of these changes is that theRyR2 channels are “leaky”. These channel leaks can result in a depletionof intracellular stores of calcium to such an extent that there is nolonger enough calcium in the sarcoplasmic reticulum (SR) to provide astrong stimulus for muscle contraction. This results in weak contractionof heart muscle. As a second consequence of the channel leaks, RyR2channels release calcium during the resting phase of the heart cycleknown as “diastole”. This release of calcium during diastole can triggerthe fatal arrhythmias of the hearts (e.g., ventricular tachycardia andventricular fibrillation) that cause sudden cardiac death (SCD).

The inventors have also shown that treatment of heart failure with amechanical pumping device, referred to as a left ventricular assistdevice (LVAD), which puts the heart at rest and restores normalizedfunction, is associated with a reduction in the PKA hyperphosphorylationof RyR2, and normalized function of the channel. Furthermore, theinventors have shown that treatment of dogs (who have pacing-inducedheart failure) with beta-adrenergic blockers (beta blockers) reversesthe PKA hyperphosphorylation of RyR2. Beta blockers inhibit the pathwaythat activates PKA. The conclusion which may be drawn from the resultsof the inventors' work is that PKA phosphorylation of RyR2 increases theactivity of the channel, resulting in the release of more calcium intothe cell for a given trigger (activator) of the channel.

As further disclosed herein, the inventors have established thatexercise-induced sudden cardiac death is associated with an increase inphosphorylation of RyR2 proteins (particularly CPVT-associated RyR2mutant proteins) and a decrease in the level of RyR2-bound FKBP12.6. Itis possible to use this mechanism to design effective drugs forpreventing exercise-induced sudden cardiac death. A candidate agenthaving the ability to limit or prevent a decrease in the level ofRyR2-bound FKBP12.6 may, as a consequence of this limiting or preventiveactivity, have an effect on an RyR2-associated biological event, therebypreventing exercise-induced sudden cardiac death.

Accordingly, the present invention further provides a method foridentifying an agent for use in preventing exercise-induced suddencardiac death. The method comprises the steps of: (a) obtaining orgenerating a culture of cells containing RyR2; (b) contacting the cellswith a candidate agent; (c) exposing the cells to one or more conditionsknown to increase phosphorylation of RyR2 in cells; and (d) determiningif the agent limits or prevents a decrease in the level of RyR2-boundFKBP12.6 in the cells. As used herein, an “agent” shall include aprotein, polypeptide, peptide, nucleic acid (including DNA or RNA),antibody, Fab fragment, F(ab′)₂ fragment, molecule, compound,antibiotic, drug, and any combination(s) thereof. An agent that limitsor prevents a decrease in the level of RyR2-bound FKBP12.6 may be eithernatural or synthetic, and may be an agent reactive with (i.e., an agentthat has affinity for, binds to, or is directed against) RyR2 and/orFKBP12.6. As further used herein, a cell “containing RyR2” is a cell(preferably, a cardiac muscle cell) in which RyR2, or a derivative orhomologue thereof, is naturally expressed or naturally occurs.

Conditions known to increase phosphorylation of RyR2 in cells include,without limitation, PKA.

In the method of the present invention, cells may be contacted with acandidate agent by any of the standard methods of effecting contactbetween drugs/agents and cells, including any modes of introduction andadministration described herein. The level of RyR2-bound FKBP12.6 in thecell may be measured or detected by known procedures, including any ofthe methods, molecular procedures, and assays known to one of skill inthe art or described herein. In one embodiment of the present invention,the agent limits or prevents a decrease in the level of RyR2-boundFKBP12.6 in the cells.

As disclosed herein, RyR2 has been implicated in a number of biologicalevents in striated muscle cells. For example, it has been shown thatRyR2 channels play an important role in EC coupling and contractility incardiac muscle cells. Therefore, it is clear that preventive drugsdesigned to limit or prevent a decrease in the level of RyR2-boundFKBP12.6 in cells, particularly cardiac muscle cells, may be useful inthe regulation of a number of RyR2-associated biological events,including EC coupling and contractility. Thus, once the candidate agentof the present invention has been screened, and has been determined tohave a suitable limiting or preventive effect on decreasing levels ofRyR2-bound FKBP12.6, it may be evaluated for its effect on EC couplingand contractility in cells, particularly cardiac muscle cells. It isexpected that the preventive agent of the present invention will beuseful for preventing exercise-induced sudden cardiac death.

Accordingly, the method of the present invention may further comprisethe steps of: (e) contacting the candidate agent with a culture of cellscontaining RyR2; and (f) determining if the agent has an effect on anRyR2-associated biological event in the cells. As used herein, an“RyR2-associated biological event” includes a biochemical orphysiological process in which RyR2 levels or activity have beenimplicated. As disclosed herein, examples of RyR2-associated biologicalevents include, without limitation, EC coupling and contractility incardiac muscle cells. According to this method of the present invention,a candidate agent may be contacted with one or more cells (preferably,cardiac muscle cells) in vitro. For example, a culture of the cells maybe incubated with a preparation containing the candidate agent. Thecandidate agent's effect on an RyR2-associated biological event then maybe assessed by any biological assays or methods known in the art,including immunoblotting, single-channel recordings and any othersdisclosed herein.

The present invention is further directed to an agent identified by theabove-described identification method, as well as a pharmaceuticalcomposition comprising the agent and a pharmaceutically-acceptablecarrier. The agent may be useful for preventing exercise-induced suddencardiac death in a subject, and for treating or preventing otherRyR2-associated conditions. As used herein, an “RyR2-associatedcondition” is a condition, disease, or disorder in which RyR2 level oractivity has been implicated, and includes an RyR2-associated biologicalevent. The RyR2-associated condition may be treated or prevented in thesubject by administering to the subject an amount of the agent effectiveto treat or prevent the RyR2-associated condition in the subject. Thisamount may be readily determined by one skilled in the art. In oneembodiment, the present invention provides a method for preventingexercise-induced sudden cardiac death in a subject, by administering theagent to the subject in an amount effective to prevent theexercise-induced sudden cardiac death in the subject.

The present invention also provides an in vivo method for identifying anagent for use in preventing exercise-induced sudden cardiac death. Themethod comprises the steps of: (a) obtaining or generating an animalcontaining RyR2; (b) administering a candidate agent to the animal; (c)exposing the animal to one or more conditions known to increasephosphorylation of RyR2 in cells; and (d) determining if the agentlimits or prevents a decrease in the level of RyR2-bound FKBP12.6 in theanimal. The method may further comprise the steps of: (e) administeringthe agent to an animal containing RyR2; and (f) determining if the agenthas an effect on an RyR2-associated biological event in the animal. Alsoprovided is an agent identified by this method; a pharmaceuticalcomposition comprising this agent; and a method for preventingexercise-induced sudden cardiac death in a subject, by administeringthis agent to the subject in an amount effective to prevent theexercise-induced sudden cardiac death in the subject.

The inventors' work has demonstrated that compounds which block PKAactivation would be expected to reduce the activation of the RyR2channel, resulting in less release of calcium into the cell. Compoundsthat bind to the RyR2 channel at the FKBP12.6 binding site, but do notcome off the channel when the channel is phosphorylated by PKA, wouldalso be expected to decrease the activity of the channel in response toPKA activation or other triggers that activate the RyR2 channel. Suchcompounds would also result in less calcium release into the cell. Inview of these findings, the present invention further providesadditional assays for identifying agents that may be useful inpreventing exercise-induced sudden cardiac death, in that they block orinhibit activation of RyR2.

By way of example, the diagnostic assays of the present invention mayscreen for the release of calcium into cells via the RyR2 channel, usingcalcium-sensitive fluorescent dyes (e.g., Fluo-3, Fura-2, and the like).Cells may be loaded with the fluorescent dye of choice, then stimulatedwith RyR2 activators to determine whether or not compounds added to thecell reduce the calcium-dependent fluorescent signal (Brillantes et al.,Stabilization of calcium release channel (ryanodine receptor) functionby FK506-binding protein. Cell, 77:513-23, 1994; Gillo et al., Calciumentry during induced differentiation in murine erythroleukemia cells.Blood, 81:783-92, 1993; Jayaraman et al., Regulation of the inositol1,4,5-trisphosphate receptor by tyrosine phosphorylation. Science,272:1492-94, 1996). Calcium-dependent fluorescent signals may bemonitored with a photomultiplier tube, and analyzed with appropriatesoftware, as previously described (Brillantes et al., Stabilization ofcalcium release channel (ryanodine receptor) function by FK506-bindingprotein. Cell, 77:513-23, 1994; Gillo et al., Calcium entry duringinduced differentiation in murine erythroleukemia cells. Blood,81:783-92, 1993; Jayaraman et al., Regulation of the inositol1,4,5-trisphosphate receptor by tyrosine phosphorylation. Science,272:1492-94, 1996). This assay can easily be automated to screen largenumbers of compounds using multiwell dishes.

To identify compounds that inhibit the PKA-dependent activation ofRyR2-mediated intracellular calcium release, an assay may involve theexpression of recombinant RyR2 channels in a heterologous expressionsystem, such as Sf9, HEK293, or CHO cells (Brillantes et al.,Stabilization of calcium release channel (ryanodine receptor) functionby FK506-binding protein. Cell, 77:513-23, 1994). RyR2 could also beco-expressed with beta-adrenergic receptors. This would permitassessment of the effect of compounds on RyR2 activation, in response toaddition of beta-adrenergic receptor agonists.

The level of PKA phosphorylation of RyR2 which correlates with thedegree of heart failure may also be assayed, and then used to determinethe efficacy of compounds designed to block the PKA phosphorylation ofthe RyR2 channel. Such an assay may be based on the use of antibodiesthat are specific for the RyR2 protein. For example, the RyR2-channelprotein may be immunoprecipitated, and then back-phosphorylated with PKAand [gamma³²P]-ATP. The amount of radioactive [³²P] label that istransferred to the RyR2 protein may be then measured using aphosphorimager (Marx et al., PKA phosphorylation dissociates FKBP12.6from the calcium release channel (ryanodine receptor): defectiveregulation in failing hearts. Cell, 101:365-76, 2000).

Another assay of the present invention involves use of aphosphoepitope-specific antibody that detects RyR2 that is PKAphosphorylated on Ser 2809. Immunoblotting with such an antibody can beused to assess efficacy of therapy for heart failure and cardiacarrhythmias. Additionally, RyR2 S2809A and RyR2 S2809D knock-in mice maybe used to assess efficacy of therapy for heart failure and cardiacarrhythmias. Such mice further provide evidence that PKAhyperphosphorylation of RyR2 is a contributing factor in heart failureand cardiac arrhythmias, by showing that the RyR2 S2809A mutationinhibits heart failure and arrhythmias, and that the RyR2 S2809Dmutation worsens heart failure and arrhythmias.

Novel Compounds, Methods of Synthesizing Same, and Uses of Same

1,4-benzothiazepine derivatives, particularly2,3,4,5-tetrahydro-1,4-benzothiazepine derivatives, are importantbuilding blocks in the preparation of biologically-active molecules,including JTV-519. The inventors have developed a novel process forpreparing 1,4-benzothiazepine intermediate compounds, such as7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine. The inventors' processutilizes readily-available and inexpensive starting materials, andprovides high yields of key 1,4-benzothiazepine intermediates.

In the early 1990s, Kaneko et al. (U.S. Pat. No. 5,416,066; WO 92/12148;JP4230681) disclosed that JTV-519 could be prepared by reacting7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (a 1,4-benzothiazepineintermediate) with acryloyl chloride, and then reacting the resultingproduct with 4-benzyl piperidine.

Two processes for the preparation of7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine and similar compoundshave been previously reported in the literature. The first process,disclosed by Kaneko et al. (U.S. Pat. No. 5,416,066), involved asynthetic route of six steps that started with 2,5-dihydroxybenzoicacid. In this process, 2,5-dihydroxybenzoic acid was selectivelymethylated with dimethyl sulfate. The resulting compound was thenreacted with dimethylthiocarbamoyl chloride for 20 h, and then subjectedto high temperature (270° C.) for 9 h. The product of this step wasrefluxed with sodium methoxide in methanol for 20 h. The product of thereflux step was then reacted with 2-chloroethylamine, under basicconditions and at a high temperature, to produce a cyclized amide. Thecyclized amide was reduced with LiAlH₄ to yield7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (a 1,4-benzothiazepineintermediate).

The second process for the preparation of7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine was disclosed byHitoshi in a Japanese patent (JP 10045706). This process started with2-bromo-5-methoxy benzaldehyde. The bromide was substituted with NaSMe,and the resulting product was oxidized with chlorine, followed by refluxin water, to yield disulfide dialdehyde. The dialdehyde was treated with2-chloroethylamine, and the resulting product was reduced with areducing agent, such as NaBH₄. The resulting compound was cyclized togive 7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine.

Initially, the inventors attempted to prepare the 1,4-benzothiazepineintermediate, 7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine, usingthe methods described above. However, they found that the first process,described by Kaneko et al. (U.S. Pat. No. 5,416,066), involved syntheticsteps of high temperature and long reaction time.

Additionally, the inventors discovered that the thio group in the thirdthiolated intermediate was easily oxidized by air to a disulfidecompound, making it impossible to synthesize the subsequent cyclizedproduct. The inventors also determined that the process described byHitoshi (JP 10045706) involved Cl₂, and that another patented method forthe preparation of the first intermediate, apart from the substitutionof bromide with NaSMe, had to be used.

To overcome the foregoing problems, the inventors developed a novelprocess for making 7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine fromreadily-available and inexpensive starting materials. The inventors'process simplifies isolation and purification steps, and can be used toprepare various 1,4-benzothiazepine intermediates, including7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine and other compoundshaving the general structure shown in formula:

This process may also be used to prepare JTV-519.

Accordingly, in view of the foregoing, the present invention provides amethod for the synthesis of a compound having formula:

wherein R=OR′, SR′, NR′, alkyl, or halide and R′=alkyl, aryl, or H, andwherein R can be at position 2, 3, 4, or 5, said method comprising thesteps of:

-   (a) treating a compound having formula:    wherein R is as defined above, with a reducing agent, in the    presence of an optional catalyst, to form a compound having formula:    wherein R is as defined above;-   (b) treating the compound formed in step (a) with a diazotizing    agent and a disulfide, to form a compound having formula:    wherein R is as defined above;-   (c) treating the compound formed in step (b) with an activating    agent and chloroethylamine, to form a compound having formula:    wherein R is as defined above;-   (d) treating the compound formed in step (c) with a reducing agent    and a base, to form a compound having formula:    wherein R is as defined above; and-   (e) treating the compound formed in step (d) with a reducing agent,    to form a compound having formula:    wherein R is as defined above.

In accordance with the method of the present invention, the reducingagent in step (a) may be H₂. Additionally, the diazotizing agent in step(b) may be NaNO₂, and the disulfide in step (b) may be Na₂S₂.Furthermore, the chloride in step (c) may be SOCl₂. The reducing agentin step (d) may be trimethylphosphine (PMe₃), while the base in step (d)is triethyl amine. In another embodiment, the reducing agent in step (e)is LiAlH₄.

The present invention further provides a method for the synthesis of acompound of having formula:

wherein R=OR′, SR′, NR′, alkyl, or halide and R′=alkyl, aryl, or H, andwherein R can be at position 2, 3, 4, or 5, said method comprising thestep of:

-   (a) treating a compound having formula:    wherein R is as defined above, with 3-bromopropionic chloride and a    compound having formula:    to form a compound having formula:    wherein R is as defined above.

By way of example, a compound having the formula:

wherein R=OR′, SR′, NR′, alkyl, or halide and R′=alkyl, aryl, or H, andwherein R can be at position 2, 3, 4, or 5, may be synthesized asfollows:

By way of example, and as shown in Example 7 and Scheme 1 below,7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine may be prepared from2-nitro-5-methoxybenzoic acid as follows. The nitro group of2-nitro-5-methoxybenzoic acid is reduced, using H₂ with Pd/C as acatalyst, to give 2-amino-5-methoxybenzoic acid.2-amino-5-methoxybenzoic acid may be diazotized with NaNO₂, and thentreated with Na₂S₂, to provide a stable disulfide compound. Withoutfurther purification, the stable disulfide compound may be treated withSOCl₂, and then reacted with 2-chloroethylamine, in the presence ofEt₃N, to give an amide. The amide compound may then be converted to acyclized compound via a one-pot procedure, as follows. A reducingreagent (such as trimethylphosphine or triphenylphosphine) and a base(such as triethylamine) may be added to a solution of the amide compoundin THF (tetrahydrofuran). The resulting reaction mixture may then berefluxed for 3 h. The reducing agent (trimethylphosphine ortriphenylphine) cleaves the disulfide (S—S) to its monosulfide (—S),which, in situ, undergoes intramolecular cyclization with the chlorideto yield a cyclized amide. The cyclized amide may then be reduced withLiAlH₄ to yield the 1,4-benzothiazepine intermediate,7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine. JTV-519 may then beprepared from 7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine byreacting the 7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine with3-bromopropionic chloride, and then reacting the resulting compound with4-benzyl piperidine.

The present invention further provides a composition, comprisingradio-labeled JTV-519. Labeling of JTV-519 may be accomplished using oneof a variety of different radioactive labels known in the art. Theradioactive label of the present invention may be, for example, aradioisotope. The radioisotope may be any isotope that emits detectableradiation, including, without limitation, ³⁵S, ¹²⁵I, ³H, or ¹⁴C.Radioactivity emitted by the radioisotope can be detected by techniqueswell known in the art. For example, gamma emission from the radioisotopemay be detected using gamma imaging techniques, particularlyscintigraphic imaging.

By way of example, and as shown in Example 8 and Scheme 2 below,radio-labeled JTV-519 may be prepared as follows. JTV-519 may bedemethylated at the phenyl ring using BBr₃. The resulting phenolcompound may then be re-methylated with a radio-labeled methylatingagent (such as ³H-dimethyl sulfate) in the presence of a base (such asNaH) to provide ³H-labeled JTV-519.

The present invention further provides novel 1,4-benzothiazepineintermediates and derivatives, including2,3,4,5-tetrahydro-1,4-benzothiazepenes that are similar to JTV-519. Byway of example, the present invention provides compounds having thefollowing formulas:

wherein R=aryl, alkenyl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, andn=0, 1, 2, or 3, and R′=alkyl or cycloalkyl;

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, and n=0, 1, 2,or 3, and R′=alkyl or cycloalkyl;

wherein R=CO(CH₂)_(n)XR′₂, SO₂(CH₂)_(n)XR′₂, or SO₂NH(CH₂)_(n)XR′₂, andX=N or S, and n=1, 2, or 3, and R′=alkyl or cycloalkyl; and wherein m=1or 2; and

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, —(CH₂)_(n)SR′, and n=0, 1, 2, or3, and R′=alkyl or cycloalkyl; and wherein X=NH or O. Also provided areadditional 2, 3, 4, 5-tetrahydro-1,4-benzothiazepine compounds havingformula:

wherein R₁=OR′, SR′, NR′, alkyl, or halide, at position 2, 3, 4, or 5 onthe phenyl ring, and R′=alkyl, aryl, or H; wherein R₂=H, alkyl, or aryl;and wherein R₃=H, alkyl, or aryl.

Examples of the inventors' novel 1,4-benzothiazepine compounds include,without limitation, S7, S-20, S-25, S-27, and S36. Preferably, thecompound is S36.

Structures for S7, S-20, S-25, S-27, and S36 may be found in Appendix A,at the end of the present application. These and any other novelcompounds of the present invention may be associated with apharmaceutically-acceptable carrier, as described above, so as to form apharmaceutical composition.

The inventors' novel 1,4-benzothiazepine compounds share functionalcharacteristics with JTV-519. For example, like JTV-519 (mwt=423),compound S36 (mwt=267) regulates calcium channels. Indeed, S36 (acarboxylic acid) is approximately 10 times more potent than JTV-519 inregulating calcium channels (data not shown). Unlike JTV-519, however,the inventors' novel compounds show weak blocking activity of hERGs.

The rapid delayed rectifier (I(Kr)) channel—a potassium channel—isimportant for repolarization of the cardiac action potential. hERG isthe pore-forming subunit of the I(Kr) channel. Suppression of I(Kr)function—as a result of adverse drug effects and/or genetic defects inhERG—can lead to long-QT (LQT) syndrome, which carries increased risk oflife-threatening arrhythmias. hERGs, then, are potassium-channelsubunits that, when blocked, can cause cardiac arrhythmias and suddencardiac death.

The inventors' compounds have significantly reduced blocking of hERG(I(Kr)) channels, when compared with JTV-519. As shown in FIGS. 4-7, forexample, one of the inventors' compounds, S36, has hERG blockingactivity that is approximately 5- to 10-fold lower than the hERGblocking activity of JTV-519. Because the inventors' compounds have weakhERG blocking activity, they are expected to be less toxic than JTV-519.

Based upon the foregoing, the inventors' novel compounds are more potentthan JTV-519, and have reduced toxicity. Accordingly, it is believedthat the inventors' novel compounds will be particularly useful in anyof the above-described methods for limiting or preventing a decrease inthe level of RyR2-bound FKBP12.6 in a subject, including a subject whois a candidate for heart failure, atrial fibrillation, orexercise-induced cardiac arrhythmia. It is also believed that theinventors' novel compounds will be particularly useful in methods fortreating or preventing heart failure, atrial fibrillation, andexercise-induced cardiac arrhythmia in a subject, and in methods forpreventing exercise-induced sudden cardiac death in a subject.

Accordingly, the present invention provides a method for limiting orpreventing a decrease in the level of RyR2-bound FKBP12.6 in a subject,comprising administering to the subject an amount of agent effective tolimit or prevent a decrease in the level of RyR2-bound FKBP12.6 in thesubject, wherein the agent is selected from the group consisting of:

wherein R=aryl, alkenyl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, andn=0, 1, 2, or 3, and R′=alkyl or cycloalkyl;

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, and n=0, 1, 2,or 3, and R′=alkyl or cycloalkyl;

wherein R=CO(CH₂)_(n)XR′₂, SO₂(CH₂)_(n)XR′₂, or SO₂NH(CH₂)_(n)XR′₂, andX=N or S, and n=1, 2, or 3, and R′=alkyl or cycloalkyl; and wherein m=1or 2;

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, —(CH₂)_(n)SR′, and n=0, 1, 2, or3, and R′=alkyl or cycloalkyl; and wherein X=NH or O; and

wherein R₁=OR′, SR′, NR′, alkyl, or halide, at position 2, 3, 4, or 5 onthe phenyl ring, and R′=alkyl, aryl, or H; wherein R₂=H, alkyl, or aryl;and wherein R₃=H, alkyl, or aryl. As described above, the subject may beany animal, but is preferably a human. In one embodiment, the subjecthas catecholaminergic polymorphic ventricular tachycardia (CPVT).

In another embodiment, the subject is a candidate for heart failure,atrial fibrillation, or exercise-induced cardiac arrhythmia. In stillanother embodiment, the agent is selected from the group consisting ofS4, S7, S-20, S-24, S-25, S-26, S-27, and S36. Structures for theseagents may be found in Appendix A herein.

In accordance with the method of the present invention, the decrease inthe level of RyR2-bound FKBP12.6 may be limited or prevented in thesubject by decreasing the level of phosphorylated RyR2 in the subject.In one embodiment, the amount of the agent effective to limit or preventa decrease in the level of RyR2-bound FKBP12.6 in the subject is anamount of the agent effective to treat or prevent heart failure, atrialfibrillation, and/or exercise-induced cardiac arrhythmia in the subject.In another embodiment, the amount of the agent effective to limit orprevent a decrease in the level of RyR2-bound FKBP12.6 in the subject isan amount of the agent effective to prevent exercise-induced suddencardiac death in the subject.

In view of the foregoing, the present invention further provides amethod for treating or preventing exercise-induced cardiac arrhythmia ina subject, comprising administering to the subject a novel1,4-benzothiazepine compound, as disclosed herein, in an amounteffective to treat or prevent exercise-induced cardiac arrhythmia in thesubject. Similarly, the present invention provides a method forpreventing exercise-induced sudden cardiac death in a subject,comprising administering to the subject a novel 1,4-benzothiazepinecompound, as disclosed herein, in an amount effective to preventexercise-induced sudden cardiac death in the subject. Additionally, thepresent invention provides a method for treating or preventing atrialfibrillation or heart failure in a subject, comprising administering tothe subject a novel 1,4-benzothiazepine compound, as disclosed herein,in an amount effective to treat or prevent the atrial fibrillation orheart failure in the subject. In each of these methods, the novel1,4-benzothiazepine compound may be selected from the group consistingof:

wherein R=aryl, alkenyl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, andn=0, 1, 2, or 3, and R′=alkyl or cycloalkyl;

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, and n=0, 1, 2,or 3, and R′=alkyl or cycloalkyl;

wherein R=CO(CH₂)_(n)XR′₂, SO₂(CH₂)_(n)XR′₂, or SO₂NH(CH₂)_(n)XR′₂, andX=N or S, and n=1, 2, or 3, and R′=alkyl or cycloalkyl; and wherein m=1or 2;

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, —(CH₂)_(n)SR′, and n=0, 1, 2, or3, and R′=alkyl or cycloalkyl; and wherein X=NH or O; and

wherein R₁=OR′, SR′, NR′, alkyl, or halide, at position 2, 3, 4, or 5 onthe phenyl ring, and R′=alkyl, aryl, or H; wherein R₂=H, alkyl, or aryl;and wherein R₃=H, alkyl, or aryl.

The present invention further provides methods of synthesizing the novel1,4-benzothiazepine compounds disclosed herein. For example, the presentinvention provides a method for the synthesis of a compound havingformula:

wherein R=aryl, alkenyl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, andn=0, 1, 2, or 3, and R′=alkyl or cycloalkyl, comprising the steps of:

-   -   (a) treating a compound having formula:    -    with a sulfonyl chloride compound (including any sulfonyl        chloride derivative) and a base, to form a compound having the        formula:    -   (b) optionally, treating the compound formed in step (a) with a        primary or secondary amine, to form a compound having formula:    -    wherein R is as defined above. In one embodiment, the sulfonyl        chloride compound in step (a) is selected from the group        consisting of alkylsulfonyl chloride and arylsulfonyl chloride.        In another embodiment, the base in step (a) is Et₃N. In still        another embodiment, the primary or secondary amine in step (b)        is 4-benzylpiperidine.

The method of the invention may further comprise the step of oxidizingthe compound having formula:

wherein R=aryl, alkenyl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, andn=0, 1, 2, or 3, and R′=alkyl or cycloalkyl, with an oxidizing agent, toform a compound having formula:

wherein R is as defined above, and wherein m=1 or 2. In one embodimentof the present invention, the oxidizing agent is hydrogen peroxide.

By way of example, and as shown in Example 9 and Scheme 3, the inventorshave developed a method of synthesizing compounds having the generalstructure:

wherein R=aryl, alkenyl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, andn=0, 1, 2, or 3, and R′=alkyl or cycloalkyl. Novel compounds of thisgeneral structure may be prepared by reacting7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine with alkylsulfonylchloride or arylsulfonyl chloride, in the presence of a base such asEt₃N. Additional reactions (e.g., addition of 4-benzyl piperidine) mayfollow, to extend the side chain as desired. As Scheme 3 demonstrates,2-chloroethanesulfonyl chloride (e.g., 180 mg; 1.1 mM) and Et₃N (e.g.,140 mg; 1.1 mM) may be added to7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (1) (e.g., 194 mg; 1mM) in CH₂Cl₂ (e.g., 20 ml), at 0° C. The mixture may then be stirred(e.g., at 0° C. for 2 h), and washed (e.g., with H₂O and saturatedNaHCO₃ solution). Removal of the solvent will yield a crude product,which may be purified by chromatography on silica gel. The structure maybe confirmed by NMR. Scheme 3 further shows that the resultingcompound's side chain may be extended by reacting the compound (e.g., 28mg; 0.1 mM) with 4-benzyl piperidine (e.g., 21 mg; 0.13 mM) in CH₂Cl₂.After the reaction goes to completion, the excess amine may be removedby a base scavenger (e.g., 3-(2-succinic anhydride)propylfunctionalizedsilica gel, 0.5 g).

The present invention also provides a method for the synthesis of acompound of having formula:

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, and n=0, 1, 2,or 3, and R′=alkyl or cycloalkyl, comprising the step of treating acompound having formula:

with a sulfuryl chloride and a primary or secondary amine, in thepresence of a base, to form a compound having the formula:

wherein R is as defined above. In one embodiment of the presentinvention, the base is Et₃N. In another embodiment, the primary orsecondary amine is 1-piperonylpiperazine.

The method of the present invention may further comprise the step ofoxidizing the compound having formula:

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, and n=0, 1, 2,or 3, and R′=alkyl or cycloalkyl, to form a compound having formula:

wherein R is as defined above, and wherein m=1 or 2. In one embodiment,the oxidizing agent is hydrogen peroxide.

By way of example, and as shown in Example 9 and Scheme 4, the inventorshave developed a method of synthesizing compounds having the generalstructure:

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, and n=0, 1, 2,or 3, and R′=alkyl or cycloalkyl. Novel compounds of this generalstructure may be prepared by a one-pot reaction of7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine with sulfuryl chloride,in the presence of a base (e.g., Et₃N), followed by a primary orsecondary amine. As Scheme 4 demonstrates, sulfuryl chloride (e.g., 15.0mg; 0.111 mM) and Et₃N (e.g., 28.0 mg; 0.22 mM) may be added to7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (e.g., 19.4 mg; 0.1 mM)in CH₂Cl₂ (e.g., 20 ml), at 0° C. After stirring the mixture (e.g., for2 h at 0° C.), 1-piperonylpiperazine (e.g., 27 mg; 0.12 mM) may beadded. The mixture may be stirred for another 2 h, and then washed(e.g., with H₂O and a saturated NaHCO₃ solution). The excess amine maybe removed by addition of a base scavenger (e.g., 3-(2-succinicanhydride) propylfunctionalized silica gel, 0.2 g).

The present invention further provides a method for the synthesis of acompound of having formula:

wherein R=CO(CH₂)_(n)XR′₂, SO₂(CH₂)_(n)XR′₂, or SO₂NH(CH₂)_(n)XR′₂, andX=N or S, and n=1, 2, or 3, and R′=alkyl or cycloalkyl; and wherein m=1or 2, comprising the step of treating a compound having formula:

wherein R is as defined above, with an oxidizing agent, to form acompound having formula:

wherein R and m are as defined above. In one embodiment, the oxidizingagent is hydrogen peroxide. This method may also be used to oxidizeJTV-519.

The present invention further provides a method for the synthesis of acompound of having formula:

wherein R=CO(CH₂)_(n)XR′₂, SO₂(CH₂)_(n)XR′₂, or SO₂NH(CH₂)_(n)XR′₂, andX=N or S, and n=1, 2, or 3, and R′=alkyl or cycloalkyl; and wherein m=1or 2, comprising the step of treating a compound having formula:

with an oxidizing agent, to form a compound having formula:

wherein R and m are as defined above. In one embodiment, the oxidizingagent is hydrogen peroxide. This method may also be used to oxidizeJTV-519.

By way of example, and as shown in Example 9 and Scheme 5, the inventorshave developed a method of synthesizing compounds having the generalstructure:

wherein R=CO(CH₂)_(n)XR′₂, SO₂(CH₂)_(n)XR′₂, or SO₂NH(CH₂)_(n)XR′₂, andX=N or S, and n=1, 2, or 3, and R′=alkyl or cycloalkyl; and wherein m=1or 2. Novel compounds of this general structure may be prepared byoxidation of JTV-519, or one of the novel 1,4-benzothiazepinederivatives disclosed herein, with hydrogen peroxide. As Scheme 5 shows,the 1,4-benzothiazepine compound of interest (e.g., 21 mg; 0.05 mM) inMeOH (e.g., 5 ml) may be added to H₂O₂ (e.g., 0.1 ml, excess). Themixture may be stirred (e.g., for 2 days), and the resulting product maybe purified by chromatography on silica gel (e.g., CH₂Cl₂:MeOH=10:1).

Additionally, the present invention provides a method for the synthesisof a compound having formula:

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, and n=0, 1, 2,or 3, and R′=alkyl or cycloalkyl; and wherein X=NH or O, comprising thestep of treating a compound having formula:

with a carbonyl chloride compound, in the presence of a base, and with aprimary or secondary amine or an alcohol, to form a compound having theformula:

wherein R and X are as defined above. In one embodiment, the carbonylchloride compound is triphosgene. In another embodiment, the base isEt₃N. In yet another embodiment, the primary or secondary amine is4-benzylpiperidine.

By way of example, and as shown in Example 9 and Scheme 6, the inventorshave developed a method of synthesizing compounds having the generalstructure:

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, —(CH₂)_(n)SR′, and n=0, 1, 2, or3, and R′=alkyl or cycloalkyl; and wherein X=NH or O. Novel compounds ofthis general structure may be prepared by reacting7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine with triphosgene, inthe presence of a base (e.g., Et₃N), followed by addition of a primaryor secondary amine or an alcohol.

The present invention further provides a method for the synthesis of 2,3, 4, 5-tetrahydro-1,4-benzothiazepine compounds having formula:

wherein R₁=OR′, SR′, NR′, alkyl, or halide, at position 2, 3, 4, or 5 onthe phenyl ring, and R′=alkyl, aryl, or H; wherein R₂=H, alkyl, or aryl;and wherein R₃=H, alkyl, or aryl, comprising the steps of:

-   -   (a) treating a compound having formula:    -    wherein R₁ is as defined above, with a reducing agent, in the        presence of an optional catalyst, to form a compound having        formula:    -    wherein R₁ is as defined above;    -   (b) treating the compound formed in step (a) with a diazotizing        agent and a disulfide, to form a compound having formula:    -    wherein R₁ is as defined above;    -   (c) treating the compound formed in step (b) with an activating        agent and chloroethylamine, to form a compound having formula:    -    wherein R₁, R₂, and R₃ are as defined above;    -   (d) treating the compound formed in step (c) with a reducing        agent and a base to form a compound having formula:    -    wherein R₁, R₂, and R₃ are as defined above; and    -   (e) treating the compound formed in step (d) with a reducing        agent, to form a compound having formula:    -    wherein R₁, R₂, and R₃ are as defined above.

The present invention also provides novel assays for regular orhigh-through-put screening of biologically-active small molecules, basedupon rebinding of FKBP12.6 and RyR2. In particular, the presentinvention provides a method for identifying an agent that enhancesbinding of RyR2 and FKBP12.6, comprising the steps of: (a) obtaining orgenerating a source of RyR2; (b) exposing the RyR2 to FKBP12.6, in thepresence of a candidate agent; and (c) determining if the agent enhancesthe binding of RyR2 and FKBP12.6. In one embodiment, the RyR2 isPKA-phosphorylated. In another embodiment, the RyR2 isPKA-hyperphosphorylated. In yet another embodiment, the RyR2 isunphosphorylated.

In the method of the present invention, the RyR2 is immobilized to asolid phase, such as a plate or beads. To facilitate detection ofRyR2-FKBP12.6 binding, the FKBP12.6 may be radio-labeled (e.g., with³²S). Furthermore, enhanced binding of RyR2 and FKBP12.6 may be detectedusing an FKBP12.6-binding agent. In one embodiment, the FKBP12.6-bindingagent is an anti-FKBP12.6 antibody. The present invention also providesan agent identified by this method, as well as uses of this agent inmethods for limiting or preventing a decrease in the level of RyR2-boundFKBP12.6 in a subject; in methods for treating or preventing heartfailure, atrial fibrillation, or exercise-induced cardiac arrhythmia ina subject; and in methods for preventing exercise-induced sudden cardiacdeath in a subject.

Additionally, the present invention provides a method for identifying anagent for enhancing the binding of RyR2 and FKBP12.6, comprising thesteps of: (a) obtaining or generating a source of FKBP12.6; (b) exposingthe FKBP12.6 to RyR2, in the presence of a candidate agent; and (c)determining if the agent enhances the binding of RyR2 and FKBP12.6. Inone embodiment, the RyR2 is PKA-phosphorylated. In another embodiment,the RyR2 is PKA-hyperphosphorylated. In yet another embodiment, the RyR2is unphosphorylated.

In the method of the present invention, the FKBP12.6 is immobilized to asolid phase, such as a plate or beads. To facilitate detection ofRyR2-FKBP12.6 binding, the RyR2 may be radio-labeled (e.g., with ³²P).Furthermore, enhanced binding of RyR2 and FKBP12.6 may be detected usingan RyR2-binding agent. In one embodiment, the RyR2-binding agent is ananti-RyR2 antibody. The present invention also provides an agentidentified by this method, as well as uses of this agent in methods forlimiting or preventing a decrease in the level of RyR2-bound FKBP12.6 ina subject; in methods for treating or preventing heart failure, atrialfibrillation, or exercise-induced cardiac arrhythmia in a subject; andin methods for preventing exercise-induced sudden cardiac death in asubject.

By way of example, and as shown in Example 10 below, a highly-efficientassay for high-throughput screening for small molecules may be developedby immobilizing FKBP12.6 (e.g., wild-type FKBP12.6 or a fusion protein,such as GST-FKBP12.6) onto a 96-well plate coated with glutathione,using standard procedures. PKA-phosphorylated ryanodine receptor type 2(RyR2) may be loaded onto the FKBP12.6-coated plate, and incubated withJTV-519 analogues and other 1,4-benzothiazepene derivatives at variousconcentrations (10-100 nM) for 30 min. Thereafter, the plate may bewashed to remove the unbound RyR2, and then incubated with anti-RyR2antibody (e.g., for 30 min). The plate may be washed again to removeunbound anti-RyR2 antibody, and then treated with florescent-labeledsecondary antibody. The plate may be read by an automatic fluorescentplate reader for binding activity.

Alternatively, RyR2 may be PKA-phosphorylated in the presence of³²P-ATP. Radioactive PKA-phosphorylated RyR2 may be loaded onto anFKBP12.6-coated, 96-well plate, in the presence of JTV-519 analogues andother 1,4-benzothiazepene derivatives at various concentrations (10-100nM) for 30 min. The plate may be washed to remove the unboundradiolabeled RyR2, and then read by an automatic plate reader.PKA-phosphorylated RyR2 also may be coated to the plate, and incubatedwith ³²S-labeled FKBP12.6 in the presence of the analogues andderivatives.

The present invention is described in the following Examples, which areset forth to aid in the understanding of the invention, and should notbe construed to limit in any way the scope of the invention as definedin the claims which follow thereafter.

EXAMPLES Example 1 FKBP12.6-Deficient Mice

FKBP12.6-deficient mice were generated, as previously described (Wehrenset al., FKBP12.6 deficiency and defective calcium release channel(ryanodine receptor) function linked to exercise-induced sudden cardiacdeath. Cell, 113:829-40, 2003). Briefly, mouse genomic λ-phage clonesfor the murine orthologue of the human FK506 binding protein 12.6(FKBP12.6) were isolated from a DBA/1lacJ library, using a full-lengthmurine cDNA probe. The targeting vector was designed to delete exons 3and 4, which contain the entire coding sequences for murine FKBP12.6(Bennett et al., Identification and characterization of the murine FK506binding protein (FKBP) 12.6 gene. Mamm. Genome, 9:1069-71, 1998), byreplacing 3.5 kb of murine genomic DNA with a PGK-neo selectable marker.A 5.0-kb 5′ fragment and a 1.9-kb 3′ fragment were cloned into pJNS2, abackbone vector with PGK-neo and PGK-TK cassettes. The DBA/lacJembryonic stem (ES) cells were grown and transfected, using establishedprotocols. Targeted ES cells were first screened by Southern analysis,and 5 positive ES cell lines were analyzed by PCR to confirm homologousrecombination. Male chimeras were bred to DBA/1 lacJ females, andgermline offspring identified by brown coat color. Germline offspringwere genotyped using 5′ Southern analysis. Positive FKBP2.6^(+/−) malesand females were intercrossed, and offspring resulted in FKBP12.6^(−/−)mice at approximately 25% frequency. FKBP12.6^(−/−) mice were fertile.

All studies performed with FKBP12.6^(−/−) mice used age- and sex-matchedFKBP12.6^(+/+) mice as controls. No differences were observed betweenFKBP12.6^(−/−) mice raised on the following backgrounds: DBA/C57BL6mixed, pure DBA, and pure C57BL6.

Example 2 Telemetry Recording and Exercise Testing in Mice

FKBP12.6^(+/+) and FKBP12.6^(−/−) mice were maintained and studiedaccording to protocols approved by the Institutional Animal Care and UseCommittee of Columbia University. Mice were anaesthetized using 2.5%isoflurane inhalation anesthesia. ECG radiotelemetry recordings ofambulatory animals were obtained >7 days after intraperitonealimplantation (Data Sciences International, St. Paul, Minn.) (Wehrens etal., FKBP12.6 deficiency and defective calcium release channel(ryanodine receptor) function linked to exercise-induced sudden cardiacdeath. Cell, 113:829-40, 2003). For stress tests, mice were exercised onan inclined treadmill until exhaustion, and then intraperitoneallyinjected with epinephrine (0.5-2.0 mg/kg) (Wehrens et al., FKBP12.6deficiency and defective calcium release channel (ryanodine receptor)function linked to exercise-induced sudden cardiac death. Cell,113:829-40, 2003). Resting heart rates of ambulatory animals wereaveraged over 4 h.

Example 3 Expression of Wild-Type and RyR2-S2809D Mutants

Mutagenesis of the PKA target site on RyR2 (RyR2-S2809D) was performed,as previously described (Wehrens et al., FKBP12.6 deficiency anddefective calcium release channel (ryanodine receptor) function linkedto exercise-induced sudden cardiac death. Cell, 113:829-40, 2003).HEK293 cells were co-transfected with 20 μg of RyR2 wild-type (WT) ormutant cDNA, and with 5 μg of FKBP12.6 cDNA, using Ca²⁺ phosphateprecipitation. Vesicles containing RyR2 channels were prepared, aspreviously described (Wehrens et al., FKBP12.6 deficiency and defectivecalcium release channel (ryanodine receptor) function linked toexercise-induced sudden cardiac death. Cell, 113:829-40, 2003).

Example 4 RyR2 PKA Phosphorylation and FKBP12.6 Binding

Cardiac SR membranes were prepared, as previously described (Marx etal., PKA phosphorylation dissociates FKBP12.6 from the calcium releasechannel (ryanodine receptor): defective regulation in failing hearts.Cell, 101:365-76, 2000; Kaftan et al., Effects of rapamycin on ryanodinereceptor/Ca⁽²⁺⁾-release channels from cardiac muscle. Circ. Res.,78:990-97, 1996). ³⁵S-labelled FKBP12.6 was generated using the TNT™Quick Coupled Transcription/Translation system from Promega (Madison,Wis.). [³H] ryanodine binding was used to quantify RyR2 levels. 100 μgof microsomes were diluted in 100 μl of 10-mM imidazole buffer (pH 6.8),incubated with 250-nM (final concentration) [³⁵S]-FKBP12.6 at 37° C. for60 min, then quenched with 500 μl of ice-cold imidazole buffer. Sampleswere centrifuged at 100,000 g for 10 min, and washed three times inimidazole buffer. The amount of bound [³⁵S]-FKBP12.6 was determined byliquid scintillation counting of the pellet.

Example 5 Immunoblots

Immunoblotting of microsomes (50 μg) was performed as described, withanti-FKBP12/12.6 (1:1,000), anti-RyR-5029 (1:3,000) (Jayaraman et al.,FK506 binding protein associated with the calcium release channel(ryanodine receptor). J. Biol. Chem., 267:9474-77, 1992), oranti-phosphoRyR2-P2809 (1:5,000) for 1 h at room temperature (Reiken etal., Beta-blockers restore calcium release channel function and improvecardiac muscle performance in human heart failure. Circulation,107:2459-66, 2003). The P2809-phosphoepitope-specific anti-RyR2 antibodyis an affinity-purified polyclonal rabbit antibody, custom-made by ZymedLaboratories (San Francisco, Calif.) using the peptide,CRTRR1-(pS)-QTSQ, which corresponds to RyR2 PKA-phosphorylated atSer²⁸⁰⁹. After incubation with HRP-labeled anti-rabbit IgG (1:5,000dilution; Transduction Laboratories, Lexington, Ky.), the blots weredeveloped using ECL (Amersham Pharmacia, Piscataway, N.J.).

Example 6 Single-Channel Recordings

Single-channel recordings of native RyR2 from mouse hearts, orrecombinant RyR2, were acquired under voltage-clamp conditions at 0 mV,as previously described (Marx et al., PKA phosphorylation dissociatesFKBP12.6 from the calcium release channel (ryanodine receptor):defective regulation in failing hearts. Cell, 101:365-76, 2000).Symmetric solutions used for channel recordings were: transcompartment—HEPES, 250 mmol/L; Ba(OH)₂, 53 mmol/L (in some experiments,Ba(OH)₂ was replaced by Ca(OH)₂); pH 7.35; and cis compartment—HEPES,250 mmol/L; Tris-base, 125 mmol/L; EGTA, 1.0 mmol/L; and CaCl₂, 0.5mmol/L; pH 7.35. Unless otherwise indicated, single-channels recordingswere made in the presence of 150-nM [Ca²⁺ ] and 1.0-mM [Mg²⁺ ] in thecis compartment. Ryanodine (5 mM) was applied to the cis compartment toconfirm identity of all channels. Data were analyzed from digitizedcurrent recordings using Fetchan software (Axon Instruments, Union City,Calif.). All data are expressed as mean±SE. The unpaired Student'st-test was used for statistical comparison of mean values betweenexperiments. A value of p<0.05 was considered statistically significant.

The effects of JTV-519 on RyR2 channels are set forth in FIGS. 1-3 andTable 1 (below). As demonstrated in FIG. 3, the single-channel studiesshowed increased open probability of RyR2 following PKA phosphorylation(D), as compared to PKA phosphorylation in the presence of the specificPKA inhibitor, PKI₅₋₂₄ (C). Single-channel function was normalized inPKA-phosphorylated RyR2 incubated with FKBP12.6 in the presence ofJTV-519 (E). Amplitude histograms (right) revealed increased activityand subconductance openings in PKA-phosphorylated RyR2, but notfollowing treatment with JTV-519 and FKBP12.6. FIG. 3F shows thatincubation of PKA-phosphorylated RyR2 with FKBP12.6, in the presence ofJTV-519, shifted the Ca²⁺-dependence of RyR2 activation towards theright, making it similar to the Ca²⁺-dependence of unphosphorylatedchannels. TABLE 1 Ambulatory ECG data before, during exercise, andfollowing exercise and injection with epinephrine. SCL (ms) HR (bpm) PR(ms) QRS (ms) QT (ms) QTc (ms) Baseline FKBP12.6^(+/−) 104 ± 6  586 ± 3632 ± 1.5 9.9 ± 0.4 30 ± 1.0 29 ± 0.6 FKBP12.6^(+/−) + JTV-519 99 ± 5 608± 32 33 ± 0.6 9.3 ± 0.3 32 ± 2.7 32 ± 1.9 FKBP12.6^(+/−) + JTV-519  116±9  527 ± 43 33 ± 0.4 10.0 ± 0.3  33 ± 1.3 30 ± 1.1 Maximum exerciseFKBP12.6^(+/−) 80 ± 2 752 ± 18 28 ± 0.7 8.7 ± 0.4 30 ± 1.7 33 ± 1.6FKBP12.6^(+/−) + JTV-519 90 ± 7 676 ± 49 29 ± 1.8 9.6 ± 0.4 34 ± 2.0 36± 0.9 FKBP12.6^(+/−) + JTV-519 83 ± 3 729 ± 22 29 ± 2   9.3 ± 0.3 30 ±1.2 33 ± 0.9 Post-exercise epinephrine FKBP12.6^(+/−) 94 ± 4 645 ± 28 35± 2.6 9.3 ± 0.4 33 ± 1.8 34 ± 1.9 FKBP12.6^(+/−) + JTV-519 102 ± 4  592± 21 37 ± 2.6 9.9 ± 0.6 32 ± 2.3 32 ± 1.7 FKBP12.6^(+/−) + JTV-519 103 ±4  585 ± 20 35 ± 3.8 11.1 ± 0.5  36 ± 1.2 36 ± 1.3Summary of ambulatory ECG data in FKBP12.6^(+/−) mice treated withJTV-519 (n = 8) or control (n = 6), and FKBP12.6^(+/−) mice treated withJTV-519 (n = 5).SCL = sinus cycle length;HR = heart rate;ms = millisecond;bpm = beats per minute;FKBP12.6^(+/−) = FKBP12.6 heterozygous mice;FKBP12.6^(+/−) = FKBP12.6 deficient mice

Example 7 Synthesis OF 1,4-Benzothiazepine Intermediate and JTV-519

For the in vivo experiments, the inventors required a gram quantity ofJTV-519. However, initial attempts to prepare this compound via thereported 1,4-benzothiazepine intermediate,7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (compound 6 in Scheme1, below), were unsuccessful. The thio group of this intermediate iseasily oxidized by air to a disulfide compound, which makes thesynthesis of cyclized product (5) impossible. To overcome this problem,the inventors developed a novel process that starts with thereadily-available and inexpensive 2-nitro-5-methoxybenzoic acid (1).This process is depicted in Scheme 1 below.

Reduction of the nitro group of compound (1), using H₂ with Pd/C as acatalyst, gave 2-amino-5-methoxybenzoic acid (2) in quantitative yield.Compound (2) was diazotized with NaNO₂, and then treated with Na₂S₂ toprovide the stable disulfide compound (3) with 80% yield. Withoutfurther purification, the stable disulfide (3) was treated with SOCl₂,and then reacted with 2-chloroethylamine, in the presence of Et₃N, togive an amide (4) in 90% yield. Compound (4) was converted to cyclizedcompound (5) via a one-pot procedure by reflux with trimethylphosphineand Et₃N in THF. The cyclized amide (5) was then reduced with LiAlH₄ toyield 7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (6).

JTV-519 was prepared by reacting compound (6) with 3-bromopropionicchloride, and then reacting the resulting product with 4-benzylpiperidine. The structure of JTV-519 was established by ¹H NMR.

Example 8 Synthesis of Radio-Labeled JTV-519

The inventors' novel process for synthesizing radio-labeled JTV-519 isdepicted in Scheme 2 below. To prepare radio-labeled JTV-519, JTV-519was demethylated at the phenyl ring using BBr₃, to give phenol compound(21). The phenol compound (21) was re-methylated with a radio-labeledmethylating agent (3H-dimethyl sulfate) in the presence of a base (NaH)to provide ³H-labeled JTV-519 (Scheme 2).

Example 9 Novel 1,4-Benzothiazepine Derivatives and Methods for TheirSynthesis

The inventors also developed novel 1,4-benzothiazepine derivatives foruse in treating and preventing cardiac arrhythmias. In particular, theinventors produced compounds having the following general structure:

wherein R=aryl, alkenyl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, andn=0, 1, 2, or 3; and wherein R′=alkyl or cycloalkyl. Novel compounds ofthis general structure were prepared by reacting7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine with alkylsulfonylchloride or arylsulfonyl chloride, in the presence of a base such asEt₃N. Additional reactions (e.g., addition of 4-benzyl piperidine) mayfollow, to extend the side chain as desired. A representative synthesisof this general process is depicted in Scheme 3 below.

As Scheme 3 demonstrates, 2-chloroethanesulfonyl chloride (180 mg; 1.1mM) and Et₃N (140 mg; 1.1 mM) were added to7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (1) (194 mg; 1 mM) inCH₂Cl₂ (20 ml), at 0° C. The mixture was stirred at 0° C. for 2 h, andwashed with H₂O and saturated NaHCO₃ solution. Removal of the solventgave crude product (Ia), which was purified by chromatography on silicagel (petroleum ether:ethyl acetate=3:1). The yield from this synthesiswas 280 mg, or 95%. The structure was confirmed by NMR.

Scheme 3 further shows that the side chain of compound (Ia) was extendedby reacting compound (Ia) (28 mg; 0.1 mM) with 4-benzyl piperidine (21mg; 0.13 mM) in CH₂Cl₂. After the reaction went to completion (by TLC),the excess amine was removed by a base scavenger (3-(2-succinicanhydride)propylfunctionalized silica gel, 0.5 g). ¹HNMR and HPLC showedthat the purity of product (Ib) was >98%.

Additionally, the inventors produced compounds having the followinggeneral structure:

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, and n=0, 1, 2,or 3; and wherein R′=alkyl or cycloalkyl. Novel compounds of thisgeneral structure were prepared by a one-pot reaction of7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (1) with sulfurylchloride, in the presence of a base (Et₃N), followed by a primary orsecondary amine. A representative synthesis of this general process isdepicted in Scheme 4 below.

As Scheme 4 demonstrates, sulfuryl chloride (15.0 mg; 0.111 mM) and Et₃N(28.0 mg; 0.22 mM) were added to7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (1) (19.4 mg; 0.1 mM)in CH₂Cl₂ (20 ml), at 0° C. After stirring the mixture for 2 h at 0° C.,1-piperonylpiperazine (27 mg; 0.12 mM) was added. The mixture wasstirred for another 2 h, and then washed with H₂O and a saturated NaHCO₃solution. The excess amine was removed by addition of a base scavenger(3-(2-succinic anhydride)propylfunctionalized silica gel, 0.2 g). Theyield from this synthesis was 36 mg, or 77%.

The inventors also produced compounds having the following generalstructure:

wherein R=CO(CH₂)_(n)XR′₂, SO₂(CH₂)_(n)XR′₂, or SO₂NH(CH₂)_(n)XR′₂, andX=N or S, and n=1, 2, or 3, and R′=alkyl or cycloalkyl; and wherein m=1or 2. Novel compounds of this general structure were prepared byoxidation of JTV-519, or one of the novel 1,4-benzothiazepinederivatives described above, with hydrogen peroxide. A representativesynthesis of this general process is depicted in Scheme 5 below.

As Scheme 5 shows, compound (Ib) (21 mg; 0.05 mM) in MeOH (5 ml) wasadded to H₂O₂ (0.1 ml, excess). The mixture was stirred for 2 days, andthe product III was purified by chromatography on silica gel(CH₂Cl₂:MeOH=10:1). The yield from this synthesis was 19 mg, or 91%.

Finally, the inventors produced compounds having the following generalstructure:

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, —(CH₂)_(n)SR′, and n=0, 1, 2, or3, and R′=alkyl or cycloalkyl; and wherein X=NH or O. Novel compounds ofthis general structure were prepared by reacting7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (1) with triphosgene,in the presence of a base (Et₃N), followed by addition of a primary orsecondary amine or an alcohol. A representative synthesis of thisgeneral process is depicted in Scheme 6 below.

Example 10 Assay for High-Throughput Screening

The inventors have developed assays for screening biologically-activesmall molecules. These assays are based on rebinding of FKBP12 proteinto RyR2.

A highly-efficient assay for high-throughput screening for smallmolecules may be developed by immobilization of FKBP12.6 (GST-fusionprotein) onto a 96-well plate coated with glutathione.PKA-phosphorylated ryanodine receptor type 2 (RyR2) is loaded onto theFKBP12.6-coated plate, and incubated with JTV-519 analogues at variousconcentrations (10-100 nM) for 30 min. Thereafter, the plate is washedto remove the unbound RyR2, and then incubated with anti-RyR2 antibodyfor 30 min. The plate is again washed to remove unbound anti-RyR2antibody, and then treated with florescent-labeled secondary antibody.The plate is read by an automatic fluorescent plate reader for bindingactivity.

In an alternative assay, RyR2 is PKA-phosphorylated in the presence of³²P_ATP. Radioactive PKA-phosphorylated RyR2 is loaded onto anFKBP12.6-coated, 96-well plate, in the presence of JTV-519 analogues atvarious concentrations (10-100 nM) for 30 min. The plate is washed toremove the unbound radiolabeled RyR2, and then read by an automaticplate reader.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art, from a reading of the disclosure, that variouschanges in form and detail can be made without departing from the truescope of the invention in the appended claims.

1. A method for identifying an agent that enhances binding of RyR2 and FKBP12.6, comprising the steps of: (a) obtaining or generating a source of RyR2; (b) exposing the RyR2 to FKBP12.6, in the presence of a candidate agent; and (c) determining if the agent enhances the binding of RyR2 and FKBP12.6.
 2. The method of claim 1, wherein the RyR2 is PKA-phosphorylated.
 3. The method of claim 2, wherein the RyR2 is PKA-hyperphosphorylated.
 4. The method of claim 1, wherein the RyR2 is immobilized to a solid phase.
 5. The method of claim 4, wherein the solid phase is a plate or beads.
 6. The method of claim 1, wherein the FKBP12.6 is radio-labeled.
 7. The method of claim 6, wherein the FKBP12.6 is labeled with ³²S.
 8. The method of claim 1, wherein enhanced binding of RyR2 and FKBP12.6 is detected using an FKBP12.6-binding agent.
 9. The method of claim 8, wherein the FKBP12.6-binding agent is an anti-FKBP12.6 antibody.
 10. An agent identified by the method of claim
 1. 11. A method for identifying an agent for enhancing the binding of RyR2 and FKBP12.6, comprising the steps of: (a) obtaining or generating a source of FKBP12.6; (b) exposing the FKBP12.6 to RyR2, in the presence of a candidate agent; and (c) determining if the agent enhances the binding of RyR2 and FKBP12.6.
 12. The method of claim 11, wherein FKBP12.6 is immobilized to a solid phase.
 13. The method of claim 12, wherein the solid phase is a plate or beads.
 14. The method of claim 11, wherein RyR2 is PKA-phosphorylated.
 15. The method of claim 14, wherein RyR2 is PKA-hyperphosphorylated.
 16. The method of claim 11, wherein the RyR2 is radio-labeled.
 17. The method of claim 16, wherein the RyR2 is labeled with ³²P.
 18. The method of claim 11, wherein enhanced binding of RyR2 and FKBP12.6 is detected using an RyR2-binding agent.
 19. The method of claim 18, wherein the RyR2-binding agent is an anti-RyR2 antibody.
 20. An agent identified by the method of claim
 11. 21. A method for limiting or preventing a decrease in the level of RyR2-bound FKBP12.6 in a subject, comprising administering to the subject an amount of agent effective to limit or prevent a decrease in the level of RyR2-bound FKBP12.6 in the subject, wherein the agent is selected from the group consisting of:

wherein R=aryl, alkenyl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, and n=0, 1, 2, or 3, and R′=alkyl or cycloalkyl;

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, or —(CH₂)_(n)SR′, and n=0, 1, 2, or 3, and R′=alkyl or cycloalkyl;

wherein R=CO(CH₂)_(n)XR′₂, SO₂(CH₂)_(n)XR′₂, or SO₂NH(CH₂)_(n)XR′₂, and X=N or S, and n=1, 2, or 3, and R′=alkyl or cycloalkyl; and wherein m=1 or 2; and

wherein R=aryl, alkyl, —(CH₂)_(n)NR′₂, —(CH₂)_(n)SR′, and n=0, 1, 2, or 3, and R′=alkyl or cycloalkyl; and wherein X=NH or O.
 22. The method of claim 20, wherein the decrease in the level of RyR2-bound FKBP12.6 is limited or prevented in the subject by decreasing the level of phosphorylated RyR2 in the subject.
 23. The method of claim 21, wherein the subject is a human.
 24. The method of claim 21, wherein the subject has catecholaminergic polymorphic ventricular tachycardia (CPVT).
 25. The method of claim 21, wherein the subject is a candidate for heart failure, atrial fibrillation, or exercise-induced cardiac arrhythmia.
 26. The method of claim 21, wherein the amount of the agent effective to limit or prevent a decrease in the level of RyR2-bound FKBP12.6 in the subject is an amount of the agent effective to treat or prevent heart failure or atrial fibrillation.
 27. The method of claim 21, wherein the amount of the agent effective to limit or prevent a decrease in the level of RyR2-bound FKBP12.6 in the subject is an amount of the agent effective to treat or prevent exercise-induced cardiac arrhythmia in the subject.
 28. The method of claim 21, wherein the amount of the agent effective to limit or prevent a decrease in the level of RyR2-bound FKBP12.6 in the subject is an amount of the agent effective to prevent exercise-induced sudden cardiac death in the subject. 